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HARVARD  SCHOOL  OF  TROPICAL  MEDICINE 


REPORT  OF  FIRST  EXPEDITION 
TO  SOUTH  AMERICA 

1913 

MEMBERS  OF  THE  EXPEDITION 


RICHARD  P.  STRONG 

PROFESSOR  OF  TROPICAL  MEDICINE,  HARVARD  UNIVERSITY 
MEDICAL  SCHOOL 


ERNEST  E.  TYZZER 

ASSISTANT  PROFESSOR  OF  PATHOLOGY 
HARVARD  UNIVERSITY  MEDICAL  SCHOOL 


A.  W.  SELLARDS 

ASSOCIATE  IN  TROPICAL  MEDICINE 
HARVARD  UNIVERSITY  MEDICAL  SCHOOL 


CHARLES  T.  BRUES 

ASSISTANT  PROFESSOR  OF  ECONOMIC 
ENTOMOLOGY,  HARVARD  UNIVERSITY 

J.  C.  GASTIABURU 

DIRECTOR  MUNICIPAL  LABORATORY 
OF  HYGIENE,  LIMA 


CAMBRIDGE 

HARVARD  UNIVERSITY  PRESS 

1915 


COPYKIGHT,  1915 
HABVARD  UNIVERSITY  PRESS 


TABLE  OF  CONTENTS 

I.    INTRODUCTION 

II.   GENERAL  CONSIDERATION  OF  OROYA  FEVER  AND  OF 
VERRUGA  PERUVIANA 

PAGE 

Historical  review 8 

Previous  views  regarding  etiology 10 

Generally  accepted  opinion  regarding  the  nature  of  verruga  peru- 
viana   13 

Present  investigations  .   ■. 14 

III.   OROYA  FEVER 

Distribution  and  seasonal  prevalence    . 15 

Clinical  features 17 

Blood 19 

Cases  emphasizing  special  clinical  features 21 

Pernicious  Oroya  fever  terminating  in  death 21 

Mild  case  of  Oroya  fever  complicated  with  verruga  peruviana  .    .  24 

Moderately  severe  Oroya  fever  terminating  in  recovery    ....  25 

Description  and  classification  of  parasite  of  Oroya  fever 28 

Forms  observed  in  fresh  blood  preparations 28 

Stained  and  fixed  preparations  of  blood 29 

Classification 30 

Development  within  endothelial  cells 32 

Attempts  to  infect  animals  with  the  parasite  op  Oroya  fever    ....  34 

Experiments 35 

Conclusions • 37 

Experiments  in  the  cultivation  of  microorganisms  from  blood  of  Oroya 

fever  cases 37 

Relationship  to  paratyphoid  infection 39 

Attempts  to  infect  and  observe  development  of  Bartonella  bacilliformis  in 

mosquitoes 41 

Pathology 42 

Previous  investigations 42 

Present  investigations 44 

Histopathology     49 

Present  investigations 49 

Studies  of  various  tissues  for  diagnosis 53 


vi  TABLE  OF  CONTENTS 

IV.   VERRUGA  PERUVIANA 

PAGE 

Geographical  distribution 57 

Conditions  in  the  endemic  areas 58 

Etiology 61 

Clinical  features 64 

Incubation  period 64 

Symptoms 64 

Eruption 65 

Blood 67 

Prognosis 69 

Cases  illustrating  special  features  of  the  disease     69 

Case     1.    Extensive  eruption;  miliary,  nodular  and  mulaire  lesions    69 
Case  17.    Papular,  cutaneous,  and  subcutaneous  nodular  eruption    73 

Case  19.    Extensive  nodular  and  mulaire  lesions 75 

Case    2.    Showing  only  miliary  eruption     76 

Cases  4  and  5.     Course  of  the  disease  in  children 78 

Case    9.     Eruption  upon  palms  of  hands  and  soles  of  feet     .    .     79 

Case  13.    Single  mulaire  lesion 80 

Case  14.    Verruga  complicated  with  malaria 81 

Experiments  relating  to  the  nature  of  the  virus  of  verruga  peruviana     81 

Transmission  to  animals,  cultivation,  filterability 81 

Experiments  with  Case  1 .     82 

Experiments  with  Case  16 105 

Experiments  with  Case  7     107 

Experiments  with  Case  17 110 

Experiments  with  Case  19 114 

Experiments  with  Case  21 119 

Experiments  with  Case  25 121 

Summary  of  transmission  experiments  to  animals 122 

Cultivation  and  filterability  op  the  virus  of  verruga 125 

Similarity  between  the  virus  of  verruga  and  that  of  small-pox  .    .   129 

Differential  diagnosis  of  verruga  peruviana 130 

From  framboesia 130 

From  Oroya  fever 133 

Relationship  to  paratyphoid  fever ' 135 

Relationship  to  angiofibroma  cutis  circumscriptum  contagiosum  .   136 

Pathology 137 

Gross  pathology 137 

Histology 140 

Previous  investigations 140 

Present  investigations  (human  and  animal  lesions)     ....   145 

Vaccination  and  immunity 152 

Transmission  of  verruga  peruviana  by  arthropoda 153 


TABLE  OF  CONTENTS  vii 

PAGE 

V.   ENTOMOLOGICAL  INVESTIGATIONS   AT   MATUCANA  .    .    161 
VI.   UTA 

CUNICAL   DESCRIPTION,    ETIOLOGY,    AND    PATHOLOGY 175 

VII.    GUAYAQUIL 

Sanitary  conditions 180 

Prevailing  diseases 182 

Entomological  investigations     184 

VIII.   YELLOW  FEVER 

Investigation  of  the  blood      189 

Clinical  histories 190 

Pathology 191 

IX.   LINGUATULIDA  FROM   CROCODILES 201 

X.    ACKNOWLEDGMENTS      203 

APPENDIX 

I.    POROCEPHALUS    CrOCODILI 207 

II.  Some  flies  of  the  family  Phorid^e 209 

III.  Notes  on  Perxtvian  mosquitoes 212 

IV.  Heteroptera  from  the  west  coast  of  South  America 218 

V.  List  of  bees  collected  by  the  expedition 220 


LIST    OF    ILLUSTRATIONS 


BUEXAVEXTURA,    COLOMBIA 

Fig.  1.   General  character  of  dwellings  in  the  town. 

Fig.  2.   General  character  of  dwellings  on  outskirts  of  town. 


II.  Chroxic  Lesioxs  of  the  Skix  Observed  in  South  America  .        4 

Fig.  1.   Espundia. 
Fig.  2.    Chronic  ulcerative  process. 
Fig.  3.   Phagedenic  ulceration. 
Fig.  4.   Yaws  in  Buenaventura. 

III.  City  of  Guayaquil 6 

Fig.  1.   Showing  elevation  above  river  and  open  drains. 
Fig.  2.   General  view  of  the  city. 

IV.  Oroya  Fever 26 

Fig.  1.  Parasite  of  Oroya  fever  in  fresh  blood  specimen,  show- 
ing changes  in  shape  and  position  in  the  cell  that  the 
organism  underwent  during  a  period  of  one  and  one- 
half  hours  in  which  it  was  observ^ed. 

Fig.  2.  Parasite  of  Oroya  fever  in  fresh  blood  specimen 
(bacillary  and  rounded  forms). 

V.  Parasite  of  Oroya  Fever  in  Stained  Blood  Specimens    .       28 

Figs.  1  and  2. 

VI.  Oroya  Fever 28 

Figs.  1  and  2.  Parasite  of  Oroya  fever  in  stained  blood  speci- 
men. 

Fig.  3.   Isolated  endothelial  cell. 

Fig.  4.  Section  showing  cycle  of  Bartonella  bacilliformis  in 
endothelial  ceils  of  lymphatic  gland. 

VII.  Oroya  Fever  Parasite  in  Red  Blood-cells  (Stained  Prepa- 

rations)           30 

Fig.  1.   Mild  infection. 

Figs.  2,  3,  4,  5,  6.  Severe  infection. 

Figs.  1,  2,  3,  4,  6,  =  X  1500.     Fig.  5,  =  X  2000. 

VIII.  Oroya  Fever 30 

Fig.  1.  Lymph-node.  Showing  swollen  endothelial  cells  in 
blood-vessel  wall. 

Fig.  2.  Smear  from  lymph-node.  Endothelial  cells.  Show- 
ing phase  in  multiplication  of  parasite.  Smear 
staincfi  by  Giemsa's  method. 


X  LIST  OF  ILLUSTRATIONS 

PLATE  PAGE 

IX.  Oroya  Fever,  Lymph-node 32 

Fig.  1.   Endothelial    cell    in    blood-vessel    of    lymph-node. 

Showing  parasite  in  form  of  rounded  bodies  with 

minute  chromatin  granules. 
Fig.  2.   Endothelial    cell    in    blood-vessel    of    Ijnnph-node. 

Showing  later  stage  of  parasite,  i.  e.,  rod  forms. 

X.  Oroya    Fever.       Endothelial    Cells    Showing    Various 

Stages  in  the  Development  of  the  Parasite;  early 
Rounded  Forms  later  Breaking  up  into  Rods.    .    .       34 

Fig.  1.  An  endothelial  cell  containing  rounded  bodies  with 
bluish  cytoplasm  and  small  granules  of  chromatin 
(interpreted  as  an  early  stage  in  the  development 
of  the  parasite). 

Fig.  2.  A  small  endothelial  cell  containing  a  number  of 
bluish  parasites,  each  containing  several  distinct 
granules  of  chromatin.  At  this  stage  none  appear 
to  contain  more  than  four  granules.  To  the  right 
is  a  red  blood-corpuscle  containing  three  rods,  each 
showing  a  chromatin  granule  with  denser  cytoplasm 
at  the  opposite  pole. 

Fig.  3.  A  large  endothehal  cell  distended  with  rounded 
masses,  each  containing  a  large  number  of  minute 
points  of  chromatin.  The  bodies  are  more  or  less 
uniform  in  size,  and  the  chromatin  granules  are  more 
minute  than  in  the  preceding  stages. 

Fig.  4.  An  endothelial  cell  containing  a  large  number  of 
minute  elements  which  appear  to  be  elongated  and 
.   to  contain  chromatin  granules. 

Fig.  5.  An  endothelial  cell  showing  numerous  minute  ele- 
ments which  appear  distinctly  rod-shaped  when 
isolated. 

Fig.  6.  A  mass  of  rod  forms  found  free  from  the  ceU.  In  the 
two  red  blood-corpuscles  to  the  right  are  character- 
istic rod-shaped  parasites  which  may  serve  as  a  basis 
of  comparison. 

Fig.  7.  A  cell  containing  small  bodies  most  distinct  in  the 
upper  left  hand  extremity,  each  with  a  small  number 
of  chromatin  points  representing  a  stage  similar  to 
that  in  Fig.  2. 

XL  Oroya  Fever 34 

Fig.  1.   Life    cycle    of    Lympho-haematocytozoon     parvum 

(after  Gonder). 
Fig.  2.   Ulceration  in  large  intestine. 

XII.  Oroya  Fever 48 

Fig.  1.   Spleen.      Area  of  necrosis  at  border  of  malpighian 

nodule. 
Fig.  2.   Liver.     Showing  area  of  necrosis,  central  type. 


PLATE 

XIII. 


XIV. 

XV. 

XVI. 

XVII. 
XVIII. 


XIX. 

XX. 

XXI. 

XXII. 

XXIII. 
XXIV. 


LIST  OF  ILLUSTRATIONS  xi 

PAGE 

Oroya  Fever 48 

Fig.  1.   Liver.     Stained  with  scharlach  Roth.    Showing  areas 

of  central  necrosis. 
Fig.  2.   Liver.     Showdng  area  of  necrosis.     Fatty  degenera- 
tion of  hver  cells  and  phagocytosis. 

Oroya  Fever  Liver      50 

Fig.  1.   Showing  necrosis  and  the  character  of  pigment. 
Fig.  2.   Sho^nng  endothelial  phagocytes  in  sinusoids,  enclos- 
ing eiythrocytes,  erythroblasts,  polymorphonuclear 
leukoc3'tes,  and  pigment. 

Oroya    Fever    Spleen    showing    Fibrin    Deposition    and 

Character  of  Pigment 50 

Oroya  Fever 52 

Fig.  1.   Liver.     Sho^ving  endothelial  phagocytes  in  sinusoid. 
Fig.  2.   Lymph-node.      Sho^^^ng  swollen  endothelial  cell  in 
blood-vessel  wall. 

Peru 58 

Fig.  1.   Regions  in  the  Andes  Mountains  in  which  Oroya 
fever,  verruga  peruviana,  and  malaria  are  prevalent. 
Fig.  2.   View  down  the  Rio  Rimac  from  above  Matucana, 
showing  the  town. 

Peru 58 

Fig.  L   Type  of  vegetation  on  the  lower  slopes  of  the  Canon 
close  to  the  river  along  the  trail  from  Surco  to  Matu- 
cana. 
Fig.  2.   Type  of  vegetation  covering  the  higher  slopes  of  the 

Canon  along  the  trail  from  Surco  to  Matucana. 
Fig.  3.   Oroya  Fever  Bridge  in  the  Andes  Mountains. 

Verruga  Peruviana  (From  Lumiere  Plates) 64 

Fig.  1.   Early  miharj'  eruption. 
Fig.  2.   Miharj'  eruption.     More  advanced  stage. 
Fig.  3.   Eruption.     Most  active  stage  of  disease. 
Fig.  4.   Eruption.     Beginning  to  recede. 

Verruga  Peruviana  (From  Lumiere  Plates)      64 

Fig.  1.   Nodular,  subcutaneous,  and  mulaire  lesions. 
Fig.  2.   Nodular  and  mulaire  lesions. 

Verruga  Peruviana 66 

Fig.  L   Acute  stage  of  disease. 
Fig.  2.   Advanced  mulaire  lesion. 

Verruga  Peruviana     .    .    .    = 66 

Fig.  L   Showing  nodular  and  subcutaneous  lesions. 
Fig.  2.   Showing  nodular  lesions. 

Verruga    Peruviana.       Showing    General    Distribution 

of  Nodular  Lesions 68 

Verruga  Inoculata  in  the  Monkey 122 


Xll 

PLATE 

XXV. 


XXVI. 


XXVII. 


XXVIII. 


XXIX. 


XXX. 


XXXI. 


XXXII. 


XXXIII. 


XXXIV. 


XXXV. 


LIST  OF  ILLUSTRATIONS 

PAGE 

Verruga  Peruviana.     Experimental  Lesions  produced  in 

Monkeys  by  Inoculation  of  Human  Virus     ....     122 

Verruga  Peruviana 124 

Fig.  1.   Dog's  testis.     Showing  experimental  lesion  produced 

by  the  inoculation  of  human  virus. 
Fig.  2.   Rabbit's  testis.      Showing  lesion  produced  by  the 
inoculation  of  human  virus. 

Verruga  Peruviana 144 

Fig.  1.   Section   of  skin  lesion.      Showing  proliferation  of 

angioblasts  about  vessels. 
Fig.  2.   Very  early  skin  lesion.     Showing  endothelial  prolif- 
eration about  vessels. 

Verruga  Peruviana 144 

Fig.  1.   Early  skin  lesion.     Showing  very  vascular  structure. 
Fig.  2.   Skin  lesion.      Section  stained  with  phosphotungstic 
acid  stain  to  show  vascularity. 

Verruga  Peruviana 146 

Fig.  1.   Nodule  of  skin.      Showing  islands  of  closely-placed 

angioblasts,     oedematous     connective    tissue,     and 

numerous  vessels. 
Fig.  2.   Nodule  of  skin.      Showing  islands  of  angioblasts, 

oedematous  connective  tissue,  and  numerous  vessels. 

Verruga  Peruviana 146 

Fig.  1.   Section  of  nodule  showing  particularly  mitosis. 
Fig.  2.   Section   showing   proliferation   of   angioblasts,    and 
appearance  of  fibroblasts. 

Verruga  Peruviana.     Lesions  of  the  Skin 148 

Fig.  1.   Section    stained    with    eosin    and   methylene    blue. 

X  10. 
Fig.  2.   Section  stained  with  Mallory's  connective  tissue  stain. 

X  10. 

Verruga  Peruviana.    Lesions  of  the  Skin 148 

Fig.  1,   Section  stained  to  show  connective  tissue. 
Fig.  2.   Section  showing  cellular  structure. 

164 

Fig.  1.   Phalangomyia  debilis  Dyar  and  Knab.     Full-grown 

larva,  dorsal  view. 
Fig.  2.   Phalangomyia    debilis    Dyar    and    Knab.       Pupa, 

lateral  view. 
Fig.  3.   ApMochaeta  scalaris  Lw.     Lateral  view  of  male. 

168 

Fig.  1.   Culicine  larva.     FuU-grown,  dorsal  view.     Surco. 
Fig.  2.   Simulium  sp.     Full-grown  larva.     Matucana. 

Uta 174 

Fig.  1.   Early  papule  above  right  lip.      Large  numbers  of 

Leishmania  found. 
Fig.  2.   Slightly  more  advanced  lesion  on  lip  than  No.  1. 


LIST   OF   ILLUSTRATIONS  xiii 

PLATE  PAGE 

XXXM.       Uta 174 

Fig.  1.   Early  lesions  on  mother  and  cliild,  slightly  more 

advanced  than  sho\\Ti  in  Plate  XXXV,  Fig.  2. 
Fig.  2.   Yery  advanced  lesions. 

XXXVII.     Uta    (From    Lumiere    Plates).      Healing    Stage    of    an 

Advanced  Case 176 

XXXM!II.   Uta.      Preparation  made   from   a   Lesion  in  the   Early 
Stage  of  the  Disease,   showing  Species  of  Leish- 

ALVNiA,  the  Etiological  Factor 178 

Figs.  1  and  2. 

XXXIX.      Uta 178 

Fig.  1.   Preparation  made  from  a  lesion  in  the  early  stage  of 
the  disease,  showing  species  of  Leishmania,  the  etio- 
logical factor. 
Fig.  2.   Flagellate  forms  of  Leishmania  obtained  in  culture 
from  an  Uta  case. 

XL.  Uta •    •    • 178 

Fig.  1.   Papular  lesions  on  inner  aspect  of  dog's  ears.     Ap- 
pearing  about   one   month   after   inoculation   with 
material  from  early  case  of  Uta. 
Fig.  2.   Showing  Leishmania  in  a  section  of  the  lesion. 

XLI.  Guayaquil 182 

Fig.  1.   Plague  Hospital. 

Fig.  2.   Yellow  Fever  Hospital. 

XLII.  Guayaquil 182 

Fig.  L   Homes  of  yellow  fever  immunes. 

Fig.  2.   Open  drain.     Breeding  place  of  Mosquitoes. 

XLIII.  Guayaquil 184 

Fig.  1.   Open  drain  behind  the  Yellow  Fever  Hospital. 
Fig.  2.   Breeding  places  of  yellow  fever  and  malaria  mos- 
quitoes. 

XLIV.  ••.••••. 184 

Fig.  1.   Milagro,  near  Guayaquil.     A  yellow  fever  district. 
Fig.  2,   Surco,  Peru,  where  uta,  Oroya  fever,  malaria,  and 
verruga  peruviana  are  prevalent. 

XLV.  Section  of  Bubo  in  Plague      186 

XLVI.  Liver  in  Yellow  Fever 189 

XLVIL        Ecuador 202 

Fig.  1 .   Crocodile  {Crocodilus  americanus)  shot  in  the  Guayas 

river. 
Fig.  2.   Daule  river.     Favorite  haunt  of  crocodiles. 

XLVIII.       Linguatulida  from  Lung  of  Crocodile 202 

Fig.  1.   Adult  Linguatulida.     About  X  21. 
P'ig.  2.   Cross-section  of  adult.     X  114. 
Fig.  3.   Greater  magnification  of  head.     Showing  mouth  and 
hooklets.    About  X  61. 


XIV 


LIST   OF  ILLUSTRATIONS 


Text  Figures: 


Fig.  1.  Phalangomyia  debilis. 
Fig.  2.  Phalangomyia  debilis 
Fig.  3.   Phalangomyia  debilis 


Fig.  4. 
Fig.  5. 
Fig.  6. 
Fig.  7. 
Fig.  8. 
Fig.  9. 


Antenna  of  larva 165 

Mandible  of  larva 165 

Maxilla 165 

Mental  plate  of  larva     ...  165 

Posterior  portion  of  larva     .  166 

Labium 168 

Labium 168 

Simulium  pupa,  No.  3 169 

Simulium  pupa,  No.  4 170 


Phalangomyia  debilis. 
Phalangomyia  debilis. 
Simulium  larva,  No.  1. 
Simulium  larva.  No.  2. 


Clinical  Chart 


20 


Tables  i  and  ii 


122 


THE  HAKVARD  SCHOOL  OF  TROPICAL  MEDICINE 

REPORT  OF  THE  FIRST  EXPEDITION 
TO  SOUTH  AMERICA,  1913 


INTRODUCTION 

The  first  expedition  from  the  Harvard  School  of  Tropical 
Medicine  was  undertaken  for  the  purpose  of  investigating 
certain  forms  of  tropical  disease  in  South  America,  particularly 
verruga  peruviana,  as  weU  as  with  the  idea  of  collecting 
material  to  be  used  for  the  instruction  of  students  in  the 
various  courses  of  the  School,  opened  in  1913. 

The  expedition  proceeded  first  to  Kingston,  Jamaica,  and 
from  there  to  Colon  and  Panama.  Observations  were  made 
concerning  the  prevailing  diseases  in  these  localities  and 
considerable  pathological  material  was  collected.  In  Kings- 
ton, malaria,  uncinariasis,  amoebic  and  bacillary  dysentery, 
and  filariasis  are  common  diseases.  An  obscure  condition 
known  as  "  vomiting  sickness  "  has  been  reported  as  occurring 
in  Jamaica  for  many  years.  Since  1904  it  has  been  more 
widely  discussed  in  medical  hterature.  More  recently  the 
investigations  of  Scott  and  Seidelin  have  attracted  attention 
to  it.  It  would  appear  that  while  microorganisms  have  been 
found  associated  with  this  disease,  its  etiology  is  still  not 
definitely  determined.  Further  observations  regarding  this 
condition  are  desirable. 

The  sanitary  conditions  in  the  Canal  Zone  and  the  diseases 
prevailing  there  have  been  already  widely  discussed  elsewhere, 
and  therefore  no  account  of  our  observations  made  in  these 
localities  will  be  given  in  this  report.  The  wards  of  the 
Panama  City  hospital  contain  many  patients  afflicted  with 
various  tropical  diseases,  and  in  the  government  hospital 
at  Ancon  a  large  number  of  individuals  suffering  with  malaria 
may  be  found. 

The  voyage  was  next  continued  down  the  west  coast  of 
South  America  to  Buenaventura,  Colombia.  This  city  is 
situated  upon  an  island  at  the  mouth  of  the  San  Juan  river  in 


4  HARVARD   SCHOOL  OF  TROPICAL  MEDICINE 

the  region  of  mangrove  swamps.  At  ebb  tide  extensive  mud 
flats  extend  out  from  the  banks  of  the  river  on  which  the  town 
is  built.  There  are  no  docks  and  the  ships  anchor  at  some 
distance  from  the  shore.  The  town  is  said  to  have  a  popula- 
tion of  10,000  at  the  present  time,  the  great  majority  of  which 
are  negroes.  The  town  has  no  system  of  drainage  and  the 
back  yards  of  the  houses  are  filthy  and  filled  with  rubbish. 
Mosquitoes  and  other  insects  are  very  numerous  about  the 
outskirts  of  the  town  and  in  the  mangrove  swamps.  A  large 
proportion  of  the  inhabitants  suffer  with  skin  diseases  of  which 
caraate  is  by  far  the  most  common.  Individuals  with  leprosy, 
yaws,  and  chronic  ulcerative  processes  of  the  skin  are  also 
frequently  observed.  (Plates  i  and  ii.)  Pathological  and 
entomological  material  was  also  collected  here. 

From  Buenaventura  we  proceeded  directly  to  Guayaquil, 
Ecuador.  From  a  sanitary  standpoint  Guayaquil  is  a  city  of 
considerable  importance  to  a  number  of  civilized  countries. 
It  is  the  largest  port  of  Ecuador  and  is  the  last  port  of  call  of 
many  steamers  on  the  northward  route  to  Balboa  and  the  Pan- 
ama Canal.  (Plate  iii.)  At  the  present  time  it  is  one  of  the 
most  unsanitary  cities  in  the  world.  It  is  particularly  danger- 
ous for  visitors  on  account  of  the  risk  to  which  they  are  exposed 
of  contracting  yellow  fever.  The  city  is  situated  two  and  one- 
half  degrees  south  of  the  equator,  on  the  right  bank  of  the 
Guayas  river,  about  forty  miles  from  its  mouth.  The  popu- 
lation is  estimated  at  80,000.  The  prevailing  diseases  are 
yellow  fever,  malaria,  bubonic  plague,  dysentery,  hookworm 
infection,  and  typhoid  fever.  Cases  of  yellow  fever  occurred 
almost  daily  during  our  stay  in  Guayaquil,  and  we  had  oppor- 
tunities of  examining  a  large  amount  of  clinical  material  and 
of  obtaining  at  autopsy  abundant  and  excellently  preserved 
material  for  histological  study.  The  blood  in  yellow  fever 
was  studied  with  relation  to  the  presence  of  parasites,  particu- 
larly with  reference  to  the  existence  of  the  protozoon  which 
Seidelin  has  described  under  the  name  of  Paraplasma  flavi- 
genum  as  the  cause  of  the  disease.  We  were  unable,  however, 
to  detect  any  bodies  which  suggested  a  parasitic  nature  in  the 


Fig.  1.  —  General  Character  of  Dwellings  in  the  Town. 


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P'lO.  2. — GeN?:IIAL    CHAKAfTEK    OF    DWELLINGS    ON    OUTSKIRTS   OF  ToWN. 


Plate  I.  —  Buenaventura,  Colombia. 


Fig.  1.  —  EspuNDiA. 


Fig.  2.  —  Chronic  Ulcerative 
Process. 


Fig.  -i.  —  Pha<;kukm('   I,  m'Khathj.v. 


FHi.  4. VaWS    in     liUKNAVENTUUA. 


I'l.ATK  II.  —  Chronic  lesions  of  the  skin  observed  in  South  America. 


IXTRODUCTIOX  5 

blood  in  this  disease.  Cultures  were  also  made  from  the 
blood  by  the  various  modern  methods,  but  no  visible  causative 
organism  could  be  detected.  A  more  detailed  account  of  the 
city  of  Guayaquil  will  be  found  in  the  section  relating  to  the 
sanitar}^  conditions  of  this  city  and  the  entomological  investiga- 
tions carried  on  there.  The  studies  relating  to  yellow  fever 
are  also  discussed  in  Section  viii  of  this  report. 

In  the  study  of  the  animal  parasites  harbored  by  the  croco- 
diles which  abound  upon  the  banks  of  the  rivers  near  Guaya- 
quil, a  species  of  Linguatulida  was  discovered.  A  description 
of  this  parasite  is  found  on  page  207. 

From  Guayaquil  we  proceeded  by  boat  to  Callao,  and  thence 
by  rail  to  Lima,  Peru.  In  the  latter  city  and  in  a  number  of 
mountain  towns  in  the  interior  of  Peru  a  large  amount  of  the 
work  of  the  expedition  was  carried  on.  The  diseases  particu- 
larly investigated  in  these  localities  w^ere  verruga  peruviana, 
Oroya  fever,  and  uta.  Isolation  by  mountain  ranges  together 
with  the  unusual  climatic  conditions  which  prevail  in  certain 
portions  of  Peru,  situated  as  they  are,  so  near  to  the  equator, 
undoubtedly  have  exercised  a  remarkable  influence  upon 
producing  what  is  in  some  respects  at  least  a  very  unusual 
fauna  and  flora,  and  hence  it  perhaps  is  not  surprising  that 
unusual  diseases  should  be  encountered  in  these  regions. 

From  remote  historical  times  the  inhabitants  of  Peru  have 
suffered  severely  from  a  disease  known  as  verruga  peruviana, 
which  is  characterized  by  fever,  anaemia,  and  a  nodular  erup- 
tion on  the  skin.  Hitherto  it  has  been  the  generally  accepted 
opinion  that  this  disease  in  its  severe  form  was  preceded  by  an 
initial  stage  known  as  the  Oroya  fever  stage,  or  the  fiebre  grave 
de  CarriGn.  If  the  individual  did  not  die  in  this  febrile  stage 
the  fever  abated  and  then  the  eruptive  or  verruga  stage  of  the 
disease  was  said  to  begin. 

The  cause  of  the  malady  was  hitherto  not  definitely  known, 
but  the  majority  of  investigators  have  attributed  a  bacterial 
origin  to  it.  Barton  described  a  bacillus  as  the  cause,  and 
later  observed  bodies  in  the  red  cells  which  he  believed  to  be 
protozoa;    by  other  observers  these  were  usually  thought  to 


6  HARVARD   SCHOOL  OF  TROPICAL  MEDICINE 

be  products  of  cell  degeneration.  More  recently  a  species  of 
Leishmania  or  one  of  Chlamydozoa  has  been  regarded  as  the 
cause  of  the  disease.  From  our  investigations  which  are 
recorded  in  detail  in  this  report,  it  would  appear  that  verruga 
peruviana  and  Oroya  fever  are  two  distinct  diseases;  that  the 
former  is  due  to  a  virus  which  may  be  transmitted  easily 
to  animals,  particularly  monkeys,  producing  characteristic 
lesions  in  them,  while  Oroya  fever  owes  its  origin  to  a  proto- 
zoan parasite  of  the  red  blood-corpuscles  and  endothelial  cells, 
sufficiently  distinct  from  other  protozoa  previously  described 
to  be  placed  in  a  new  genus.  To  this  parasite  we  have  given 
the  name  of  Bartonella  bacilliformis.  Also  we  have  been  able 
to  demonstrate  a  method  of  vaccination  against  verruga 
peruviana.  A  detailed  description  of  our  observations  relat- 
ing to  these  diseases  will  be  found  in  Sections  iii  and  iv. 

Uta  is  an  ulcerative  disease  which  has  been  known  in  Peru 
since  prehistoric  times.  Lesions  of  the  malady  have  been 
depicted  on  the  "huacos"  or  ancient  pottery  of  the  Incas. 
It  has  been  supposed  that  the  disease  was  a  prehistoric  form 
of  syphilis  or  one  of  leprosy.  More  recently  it  has  been 
regarded  as  a  form  of  lupus  vulgaris,  or  as  a  distinct  infection. 
Its  etiology  hitherto  had  not  been  determined.  We  were  able 
to  show  that  uta  is  due  to  a  species  of  Leishmania.  We  were 
also  able  to  successfully  obtain  the  flagellate  stage  of  the 
organism  in  cultures  and  to  inoculate  successfully  a  dog  with 
it.  The  researches  undertaken  in  connection  with  uta  will  be 
found  in  Section  vi. 

Acknowledgment  of  both  official  and  financial  assistance 
rendered  the  expedition  will  be  found  at  the  end  of  the  report. 
The  other  scientific  members  of  the  expedition  were  Doctor 
E.  E.  Tyzzer,  Assistant  Professor  of  Pathology,  C.  T.  Brues, 
Assistant  Professor  of  Economic  Entomology,  both  of  Harvard 
University,  and  Dr.  A.  W.  Sellards,  formerly  of  Johns  Hopkins 
University  and  now  Associate  in  Tropical  Medicine  at  Har- 
vard University.  In  Lima  the  services  of  Dr.  J.  C.  Gastia- 
burii.  Chief  of  the  Institute  of  Hygiene,  Lima,  were  enlisted 
in  connection  with  the  work  in  Peru.     Miss  Nora  A.  Dwyer, 


Fig.  1. — Showing  elevation  above  river  and  open  drains. 


Fio.  2.  —  General  view  ok  the  city. 
Plate  III.  —  City  of  Guayaquil. 


INTRODUCTION  7 

Secretar}^  to    President    A.   Lawrence   Lowell,  accompanied 
the  expedition  as  secretary. 

The  results  obtained  and  the  discoveries  made  by  the 
expedition  should  serve  to  emphasize  the  importance  of 
sending  from  the  School  to  the  tropics  other  expeditions  of 
this  nature  for  the  investigation  of  specific  problems.  In 
this  instance  the  additional  knowledge  obtained  relating  to 
the  diseases  studied  has  been  secured  at  a  comparatively 
trifling  cost. 


II 

GENERAL  CONSIDERATION  OF 
OROYA  FEVER  AND  VERRUGA  PERUVIANA 

Historical  Review 

From  remote  historical  times  the  inhabitants  of  Peru  are 
said  to  have  suffered  severely  from  an  obscure  disease  char- 
acterized by  fever,  anaemia,  and  a  nodular  eruption  upon  the 
skin.  Over  four  centuries  ago,  during  the  reign  of  the  Inca, 
Huayna  Capac,  thousands  of  lives  were  swept  away,  sup- 
posedly from  this  malady,  and  in  the  history  of  the  conquest 
of  Peru  by  Zarate,^  published  in  1545,  it  is  stated  that  the 
disease  is  more  destructive  than  small-pox,  and  almost  as 
disastrous  as  the  plague  itself.  Later  this  author  mentions 
that  the  Portuguese  soldiers  were  afflicted  by  boils  or  warts 
of  a  very  malignant  kind,  and  that  not  a  single  person  in  the 
army  escaped  them.  De  la  Vega  also  relates  that  during  the 
16th  century  a  quarter  of  the  invading  army  of  Peru  under 
Francisco  Pizarro  perished  from  this  disease.  In  1842  Archi- 
bald Smith  2  called  attention  again  to  the  malady,  and  shortly 
afterward  a  number  of  additional  papers  relating  to  it  were 
published  by  Tschudi,^  Odriozola,^  and  Salazar.^  Later 
Dounon  ^  in  1871,  Fournier  "'  in  1874,  Bourse  ^  and  Tupper  ^ 
in  1877,  all  described  the  disease. 

^  Zarate:  Historia  del  discubrimento  de  Peru,  1545,  i,  4;  ii,  1. 

2  Smith:  Edinb.  M.  and  S.  J.,  1842,  67. 

3  Tschudi:  Arch.  f.  physiol.  Heilk.,  Stuttg.,  1845,  iv,  378. 

Oesterr  med.  Wchnschr.,  Wien,  1846,  505. 
*  Odriozola:  Gac.  med.  de  Lima,  1858,  April. 

Med.  Times  and  Gaz.,  Lond.,  1858,  Sept.,  280. 
^  Salazar:  Thesis,  Gac.  med.  de  Lima,  1860. 
^  Dounon:  Arch,  de  med.  nav..  Par.,  1871,  255. 

These  de  Paris,  1871. 
^  Fournier:  Arch,  de  med.  nav..  Par.,  1874,  156. 
^  Bourse:  Arch,  de  med.  nav..  Par.,  1876,  353. 
^  Tupper:  Ueber  die  Verruca  peruviana,  Inaug.-Diss.,  Berl.,  1877. 


HISTORICAL   REVIEW  9 

In  1870  a  severe  outbreak  of  fever  took  place  among  the 
workmen  building  the  Central  Railway  between  Lima  and 
Oroya,  and  it  is  estimated  that  at  least  seven  thousand  indi- 
viduals died  during  the  epidemic.  At  this  time  the  com- 
plaint received  the  name  of  ''Oroya  fever/'  although  it  appears 
that  it  was  not  contracted  in  the  town  of  Oroj^a  itself. 
Bourse  reported  that  all  of  the  engineers  superintending  the 
building  of  the  Trans- Andean  Railway  contracted  verruga, 
and  that  half  of  them  died  of  it.  Of  forty  sailors  who  had 
deserted  from  a  British  ship  and  gone  to  work  on  the  railway, 
thirty  died  of  the  disease  in  the  course  of  seven  or  eight 
months.  In  1906  out  of  a  force  of  two  thousand  men  employed 
in  tunnel  w^ork  for  the  Central  Railway,  two  hundred  are 
known  to  have  died  of  the  disease.  It  appeared  to  be  only 
necessary  for  the  workmen  to  spend  a  single  night  in  the 
infected  districts  in  order  to  contract  the  malady. 

Previous  to  1885  there  ensued  some  discussion  as  to  whether 
Oroya  fever  and  verruga  peruviana  were  related  to  one  another, 
or  whether  the  latter  was  a  distinct  disease.  August  27,  1885, 
Daniel  Carrion,  a  medical  student  in  Lima,  and  a  native  of 
Cerro  de  Pasco,  Peru  (a  town  situated  in  the  mountains  far 
above  the  localities  in  which  the  disease  abounds),  attempted 
to  solve  this  problem  by  vaccinating  both  his  arms  with  blood 
from  a  verruga  nodule.  It  is  related  that  twenty-three  daj^s 
later  he  began  to  suffer  from  Oroya  fever,  from  which  he  died 
sixteen  days  later,  or  on  October  5.  From  this  experiment  the 
conclusion  was  drawn  b}^  Peruvian  physicians  that  verruga 
and  Oroya  fever  were  only  different  stages  of  the  same  disease, 
and  this  is  the  opinion  which  has  been  held  generally  by 
them  since  this  time.  In  honor  of  Carrion's  attempt  to 
throw  light  on  the  question,  the  febrile  condition,  which  has 
been  regarded  as  the  first  stage  of  the  malady,  is  now  generally 
known  in  Peru  as  "  ficbre  de  Carrion."  Although  it  has  been 
stated  that  Carrion  during  his  illness  kept  notes,  and  gave  a 
minute  description  of  his  symptoms  to  his  companions,  un- 
fortunately it  appears  that  none  of  these  were  preserved  and 
published.     No  accurate  record  of  Carrion's  case  and  of  the 


10     OROYA  FEVER  AND  VERRUGA  PERUVIANA 

necropsy  is  available.  It  has  been  suggested  since  that  he 
died  of  typhoid  fever  or  of  a  more  acute  form  of  septicemia, 
and  it  is  also  quite  possible  that  the  patient  from  which  he 
infected  himself  was  suffering  with  Oroya  fever  as  well  as 
verruga  at  the  time. 

In  1898  the  monograph  of  Ernesto  Odriozola  ^  appeared, 
which  constitutes  the  most  excellent  and  most  comprehensive 
account  of  the  disease  which  has  been  written.  To  this 
monograph  the  reader  is  referred  for  the  complete  history  of 
the  disease,  its  geographical  distribution,  and  indeed  for  all 
points  concerning  our  knowledge  of  it  and  its  cause  up  to  that 
date. 

Previous  Views  Regarding  Etiology 

If  we  turn  our  attention  to  the  studies  which  have  been 
carried  on  previously  in  relation  to  the  etiology  of  these  condi- 
tions, we  find  very  conflicting  opinions,  although  the  majority 
of  writers  on  this  subject  have  ascribed  a  bacterial  origin  to 
the  disease.  Izquierdo  ^  in  1884  reported  the  presence  of  a 
bacillus  in  the  study  of  sections  of  tissues  sent  him  from  Peru, 
and  Florez  ^  in  1887  from  the  blood  of  individuals  affected 
with  verruga  cultivated  on  agar-agar  a  coccus.  Odriozola^ 
also  reported  the  presence  of  a  bacillus  in  the  blood. 

In  1901  and  1902  Barton  ^  performed  extensive  bacterio- 
logical investigations,  and  concluded  that  in  the  blood  and 
organs  at  necropsy  of  persons  dying  from  Carrion's  fever  a 
bacillus  was  present  which,  though  similar  to  Bacillus  coli 
communis,  could  be  distinguished  from  it.  This  organism, 
which  he  regarded  as  the  cause  of  the  disease,  was  said  to  give 
rise  either  to  a  fatal  septicemia  in  animals,  or  to  a  verruga-like 
eruption  of  the  skin.     Biffi  and  Carbajal,^  and  Tamayo  and 

1  Odriozola:  La  Maladie  de  Carrion  ou  la  Verruga  Peruvienne,  Par.,  1898,  Carre 
et  Naud,  Editeurs. 

2  Izquierdo:  Virchow's  Arch.  f.  path.  Anat.,  Berl.,  1885,  xcix,  411. 
^  Florez :  Odriozola's  Monograph,  p.  100. 

^  Odriozola:  loc.  cit.,  p.  102. 

5  Barton:  Cron.  med.,  Lima,  1901,  xviii.  No.  301,  p.  193;  No.  302,  p.  210;  1902,  xix, 
No.  334,  p.  348. 

6  Biffi  and  Carbajal:  Cron.  med.,  Lima,  1904,  xxi.  No.  379,  p.  285;  1903,  xx,  No.  346, 
p.  149.     Biffi:  Arch.  f.  Schiffs-  u.  Tropenhyg.,  Leipz.,  1908,  xii,  1. 


PREVIOUS   VIEWS   REGARDING   ETIOLOGY  11 

Gastiabum  ^  investigated  this  bacillus,  and  reported  that  it 
was  present  constantly  in  persons  suffering  from  Carrion's 
fever,  and  was  agglutinated  by  their  serum,  but  was  absent 
from  persons  suffering  from  verruga  peruviana  without  fever, 
whose  serum  also  showed  no  agglutination  of  this  bacillus. 
They  identified  the  organisms  isolated  as  belonging  in  the 
paratyphoid  group.  They  were  unable  to  confirm  Barton's 
results  as  to  the  production  in  animals  of  a  skin  eruption  by 
the  bacillus,  and  concluded  that  it  may  be  a  secondary  in- 
vader in  verruga,  or  may  give  rise  to  the  symptoms  of  a  form 
of  fever  resembling  typhoid  which  constituted  the  fiebre  grave 
de  Carrion.'^  In  1903  Bifii  ^  and  Gastiaburii  ^  noted  in  stained 
preparations  of  the  blood  the  presence  of  granules  in  the  red 
blood-cells  which  stained  readily  with  the  basic  anilin  dyes. 
The  form  and  structure  of  these  bodies  was  that  of  a  cocco- 
bacillus  or  of  irregular  granules.  In  1905,  before  the  Sociedad 
IMedica  Union  Fernandina,  Barton  ^  described  in  the  red 
blood-cells  of  two  persons  sick  with  severe  malignant  fever 
elements  similar  in  morphology  to  baciUi.  In  1909  he  noted 
the  presence  of  these  elements  in  stained  blood  specimens  in 
fourteen  additional  cases  and  expressed  the  belief  that  they 
were  protozoa  and  probably  the  specific  agent  of  the  infection. 
In  1909  Gastiaburii  and  Rebaghati  ^  observed  the  bodies 
described  by  Barton  and  stated  that  they  were  probably 
protozoa  and  might  be  regarded  as  the  pathogenic  organism 
in  Carrion's  disease.  Later,  De  Vecchi,"'  Mayer,^  Gastiaburii 
and  RebagUati,^  Monge,^°  and  several  other  observers  have 

1  Tamayo  and  Gastiaburii:  Gac.  de  1.  hosp.,  1905,  ii,  No.  46,  p.  516;  Cr6n.  m6d., 
Lima,  1905,  xxii.  No.  406,  p.  335;  No.  407,  p.  349;  1906,  xxiii,  No.  429,  p.  327;  1907, 
xxiv.  No.  453,  p.  321;  Gac.  de  1.  hosp.,  1906,  iii.  No.  62,  p.  107. 

2  Biffi  believed  that  it  was  not  proved  that  verruga  peruviana  and  the  severe  fever 
of  Carrion  were  two  stages  of  one  and  the  same  disease. 

»  Biffi:  Cr6n.  m^d.,  Lima,  1903,  xx.  No.  346,  p.  149. 

♦  Gastiaburii:  Cr6n.  m6d.,  Lima,  1903,  xx,  No.  356,  p.  314. 
»  Barton:  Cr6n.  m<-d.,  Lima,  1909,  xxvi.  No.  481.  p.  7. 

•  Gastiaburii  and  Kel>agliati:  CrcOn.  m^d.,  Lima,  1909,  xxvi,  No.  501,  p.  378. 
'  De  Vecohi:  Beiheft  4,  Arch.  f.  Schiffs-  u.  Tropenhyg.,  Leipz.,  1909,  xiii,  143. 
»  Mayer:  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  Jena,  1910,  Ivi,  Orig.  309. 

»  Gastiaburii  and  Rebagliati:  Cr6n.  rn6d.,  Lima,  1912,  xxix.  No.  571,  p.  644;  No. 
572,  p.  651. 

»»  Monge:  J.  Umd.  School  Trop.  iMed.,  1912,  i.  Part  2,  163;  Cr6n.  m6d.,  Lima, 
1912,  xxix,  No.  571,  p.  640. 


12     OROYA  FEVER  AND  VERRUGA  PERUVIANA 

been  inclined  to  believe  that  the  bodies  described  by  Barton 
were  products  of  cell  degeneration.  Bassett-Smith  ^  and  Galli 
Valerio  ^  studied  several  stained  specimens  of  the  blood  sent 
them  from  Peru.  Bassett-Smith  concludes  that  before  any 
opinion  is  arrived  at  as  to  the  nature  of  the  inclusions  in  the 
red  cells,  a  detailed  study  of  a  large  number  of  fresh  specimens 
should  be  made  to  see  if  their  presence  is  constant  in  all  acute 
cases.  Galli  Valerio  mentions  that  in  the  red  cells,  he  observed 
the  bodies  previously  described,  some  of  which  in  one  speci- 
men resembled  Anaplasma  marginale  of  cattle.  He  regards 
the  etiology  of  verruga  peruviana  as  still  undetermined.  On 
the  other  hand,  Nicolle,^  LetuUe,^  and  Galli  Valerio,^  found 
acid-fast  bacilli  in  the  lesions  from  patients  with  verruga. 
Darling,*'  whose  publication  relating  to  this  disease  is  based 
on  a  necropsy  made  in  Panama  the  day  following  the  death 
of  a  patient  with  verruga  complicated  with  tuberculosis, 
suggests  that  the  acid-fast  bacilli  previously  described  were 
really  tubercle  bacilli  occurring  in  cases  complicated  by  tuber- 
culosis. In  the  same  paper  Darling  apparently  from  an 
examination  of  stained  blood  films  not  from  his  own  case, 
but  sent  him  from  Peru  by  Barton,  concludes  that  the  slender 
rod-like  bodies  in  the  red  cells  which  he  refers  to  as  '^  x 
bodies,"  seemed  to  represent  some  unique  type  of  micro- 
organism. In  his  own  case  of  verruga  no  '^  x  bodies  "  were 
observed  in  the  preparations,  with  the  possible  exception  of 
the  smears  made  from  the  rib  marrow,  which  he  states  con- 
tained a  few  rod-like  bodies,  not  definitely  in  the  erythro- 
cytes. Still  more  recently  Gastiaburu  and  Rebagliati  ^  have 
found  in  the  liver  and  skin  lesions  of  a  verruga  patient 
in  the  eruptive  phase,  certain  bodies,  at  times  endoglobular 

1  Bassett-Smith:  Brit.  M.  J.,  Lond.,  1909,  ii,  783. 

2  Valerio,  Galli:    Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  Jena,  1911,  Iviii,  Part  I, 
Orig.,  228. 

3  NicoUe:  Ann.  de  I'Inst.  Pasteur,  Par.,  1898,  xii,  591. 

*  Letulle:  Compt.  rend.  Soc.  debiol:.  Par.,  1898,  764;  Odriozola's  Monograph,  p.  201. 
^  Valerio,  Galli:  loc.  cit. 

6  Darling,  S.  T.:  J.  Am.  M.  Ass.,  Chicago,  1911,  Dec.  23,  Ivii,  2071. 
^  Gastiaburu  and  Rebagliati:   Cr6n.  med.,  Lima,  1912,  xxix.  No.  571,  p.  644;  No. 
572,  p.  651. 


PREVIOUS   VIEWS   REGARDING   ETIOLOGY  13 

fn  leukocytes  and  other  cells,  and  at  other  times  free,  which 
from  their  staining  reactions  and  morphologic  appearance 
they  regarded  as  probabl}'  organisms  of  the  genus  Leish- 
mania.  Rebaghati  also  found  certain  endogiobular  bodies 
which  he  considered  to  be  remnants  of  nuclear  disorgan- 
ization of  the  erythrocytes,  which  suggested  that  Barton's 
bodies  might  be  chromatin  filaments,  segregated  from  the 
nuclei  of  erythroblasts.  Finally,  during  the  past  year, 
]\Iayer,  Rocha-Lima,  and  H.  Werner,^  in  the  study  of  a 
single  case  of  verruga  in  Hamburg,  believed  that  they  had 
found  bodies  resembling  Chlamydozoa  in  the  cells  in  the  skin 
lesions. 

Thus  it  will  be  seen  from  this  review  regarding  the  previous 
investigations  pursued,  that  at  the  time  our  studies  in  relation 
to  this  subject  were  undertaken,  the  etiology  of  verruga  peru- 
viana and  of  Oroya  fever  was  undetermined,  and  the  question 
was  in  a  confused  condition,  owing  to  the  fact  that  the  work  of 
previous  investigators  had  not  been  confirmed. 

Generally  Accepted  Opinion  Regarding  the  Nature  of 
Verruga  Peruviana 

According  to  the  generally  accepted  opinion  among  the 
physicians  of  Peru  at  the  time  of  our  arrival  in  Lima,  the 
disease  verruga  peruviana  in  the  severe  form  begins  with  an 
initial  stage  known  as  the  fiebre  grave  de  Carrion  which  is 
characterized  by  a  fever  which  lasts  from  fifteen  to  thirty  days, 
profound  anaemia,  prostration,  and  a  high  mortahty.  If  the 
patient  does  not  die  in  this  stage  the  fever  begins  to  abate, 
and  the  eruptive,  or  verruga  stage  commences.  If  the  erup- 
tion is  generalized  and  abundant,  then  it  is  stated  that  the 
patient  is  sure  to  recover.  In  the  chronic  or  mild  form  of 
the  disease,  which  is  said  to  comi)rise  the  great  proportion  of 
the  cases,  there  is  moderate  fever  of  intermittent  or  remittent 
character,  and  pains  in  the  joints  are  common;  more  or  less 
anaemia  is  present.     The  eruption  is  said  to  be  the  culminat- 

'  Mayer,  Rocha-Lima,  H.  Werner:   Munchen.  tned.  Wchnschr.,  1913,  No.  14. 


14     OROYA  FEVER  AND  VERRUGA  PERUVIANA 

ing  feature  in  both  forms,  and  it  appears  under  various  types 
which,  according  to  the  special  characteristics  they  reveal, 
are  termed  "  miUary,"  "  nodular,"  or  '^  mulaire." 

Pkesent  Investigations 

After  studying  these  conditions  in  Peru,  we  concluded  that 
verruga  peruviana  and  Oroya  fever  are  two  distinct  diseases. 
We  have  been  able  to  show  that  the  former  is  due  to  a  virus 
which  may  be  transmitted  to  animals  by  direct  inoculation 
and  which  produces  definite  lesions  in  them,  and  that  the 
latter  is  due  to  an  organism  parasitic  in  the  red  blood-cor- 
puscles and  endothelial  cells,  and  sufficiently  distinct  from  the 
other  hematozoa  to  be  placed  in  a  new  genus.  So  far  this 
organism  has  not  been  successfully  transmitted  to  the  lower 
animals.  The  parasite  which  we  consider  to  be  the  cause  of 
Oroya  fever  produces  in  man  fever,  and  in  severe  infections  a 
rapid  and  very  pernicious  form  of  anaemia,  which  results  in 
extreme  prostration  and  frequently  in  death. 

Verruga  peruviana,  however,  is  evidently  very  rarely  a 
fatal  disease  when  uncomplicated  with  other  infections.  It  is 
particularly  characterized  by  an  eruption  upon  the  skin  which 
may  assume  a  very  different  appearance  in  different  stages  of 
the  disease.  Our  investigations  relating  to  these  two  diseases 
and  the  evidence  upon  which  these  conclusions  are  based  will 
now  be  considered  in  detail. 


Ill 

OROYA   FEVER 

Distribution  and  Seasonal  Prevalence 

Our  investigations  relating  to  Oroya  fever  and  verruga  peru- 
viana were  made  in  Lima,  in  the  large  hospitals  in  that  city, 
and  in  a  number  of  the  mountain  towns  in  the  interior  of  Peru, 
particularly  San  Mateo,  Chosica,  Santa  Eulalia,  San  Barto- 
lome,  Surco,  Cocochacra,  and  Matucana.  Cases  of  verruga 
peruviana  were  found  in  or  in  the  vicinity  of  all  of  these  towns, 
and  in  these  localities  cases  of  malaria  were  also  common. 
Cases  of  Oroya  fever  were  studied  in  which  the  disease  had 
been  contracted  in  or  in  the  vicinity  of  San  Bartolome  and 
Matucana. 

At  the  present  time  our  knowledge  of  the  geographical 
distribution  of  Oroya  fever  probably  is  very  incomplete.  The 
disease  undoubtedly  occurs  in  many  of  the  deeply  cleft,  narrow 
valleys  situated  on  the  western  slope  of  the  Peruvian  Andes 
at  altitudes  of  from  2,500  to  8,000  feet.  In  these  districts,  as 
has  been  intimated,  malaria  and  verruga  peruviana  are  also 
very  common  diseases.  Owing  to  the  fact  that  Oroya  fever, 
or  Carrion's  disease  as  it  is  frequently  termed  in  these  localities, 
evidently  has  been  confused  so  often  with  other  diseases  en- 
countered there,  and  particularly  with  verruga  peruviana, 
malaria,  and  paratyphoid  fever,  our  knowledge  of  its  distribu- 
tion even  in  these  regions  must  be  far  from  accurate.  More- 
over, it  would  not  be  surprising  if  cases  of  Oroya  fever  were 
subsequently  found  to  occur  in  other  tropical  countries.  Since 
the  parasite  of  the  disease  is  difficult  to  detect,  and  its  para- 
sitic nature  up  to  the  time  of  our  investigations  had  not  been 
definitely  established,  it  would  not  seem  strange  if  the  disease 
has  escaped  observation  in  other  localities  where  it  exists. 

Like  malaria,  the  disease  appears  undoubtedly  to  have  a 
seasonal  prevalence,  and  this  appears  to  be  more  marked  than 

16 


16  OROYA  FEVER 

in  the  case  of  verruga  peruviana.  Oroya  fever  seems  to  be 
much  more  prevalent  in  Peru  from  January  to  April,  and 
particularly  towards  the  close  of  the  warm,  rainy  season.  Ac- 
cording to  the  hospital  reports  in  Lima,  in  1913,  more  cases 
were  observed,  in  January,  February,  and  March.  In  April 
and  May  there  were  no  admissions  with  the  diagnosis  of  this 
disease  at  the  Dos  de  Mayo  hospital.  Our  investigations  in 
Peru  were  largely  carried  out  during  the  months  of  June,  July, 
and  August,  which  is  the  season  in  which  neither  Oroya  fever 
nor  malaria  are  particularly  prevalent  there.  However, 
during  these  months  verruga  cases  were  certainly  not  uncom- 
mon according  to  our  observations. 

It  is  important  to  emphasize  that  Odriozola  ^  in  that  por- 
tion of  his  monograph  which  relates  to  the  fievre  grave  de  Car- 
rion, in  discussing  the  localities  in  which  this  disease  is 
encountered  asks  the  question:  "  Pourquoi,  en  effet,  dans 
quelques  cas,  voyons-nous  apparaitre  la  fievre  grave  de  Car- 
rion, tandis  que,  dans  d'autres,  c'est  I'eruption  benigne  qui  se 
presente?  "  He  then  goes  on  to  point,  out  just  what  the 
climatic  conditions  and  physical  features  are  in  these  valleys 
where  the  severe  fievre  grave  de  Carrion  is  encountered,  and 
concludes  a  discussion  of  this  part  of  the  subject  with  the 
statement:  "  Par  contre,  les  quebradas  qui  sont  perpendi- 
culaires  a  la  cote  jouissent  d'une  certaine  ventilation:  elles 
sont  plus  ou  moins  traversees  par  les  vents  de  la  mer  et  de  la 
Sierra;  les  variations  de  temperature  y  sont  plus  frequentes,  a 
cause  de  la  plus  grande  facilite  qu'a  Tair  de  s'y  renouveler. 
Done,  dans  ces  quebradas,  c'est  la  maladie  de  Carrion  benigne, 
ou  de  moyenne  intensite,  qui  prendra  naissance;  et  c'est 
justement  ce  qui  arrive." 

These  statements  from  so  eminent  an  authority  are  of  the 
greatest  importance  for  they  seem  to  suggest  to  us  that  the 
distribution  or  rather  the  prevalence  of  Oroya  fever  and  of 
verruga  peruviana  does  not  always  coincide  in  regard  to  time 
and  place,  any  more  than  does  the  distribution  of  verruga 
peruviana  and  malaria. 

1  Odriozola:   loc.  cit.,  p.  88. 


CLINICAL  DESCRIPTION  17 

Clinical  Features 

Clinically  the  disease  is  particularly  characterized  by  a 
fever  which  may  be  irregular  in  its  course  (see  Chart  1,  page 
21),  and  in  severe  infections  by  a  rapid  and  pernicious  form 
of  anaemia  which  results  in  extreme  prostration  and  frequently 
in  death.  In  severe  infections  the  patient  may  not  live  longer 
than  three  or  four  weeks  from  the  onset  of  the  symptoms. 

Descriptions  in  the  literature  regarding  the  clinical  mani- 
festations of  this  disease  are  often  or,  indeed,  usually  confusing, 
since  almost  invariably  in  these  descriptions  Oroya  fever  and 
verruga  peruviana  are  described  as  merely  different  stages  of 
the  same  disease.  The  cases  of  Oroya  fever  which  have 
terminated  fatally  are  said  to  have  died  in  the  first  stage  of 
the  disease,  verruga,  before  the  eruption  has  appeared  upon  the 
skin.  Those  cases  of  true  verruga  in  which  an  eruption 
develops  are  said  to  be  in  the  eruptive  stage  of  the  disease. 

Concomitant  infections  with  Oroya  fever  and  verruga 
peruviana  may  occur,  as  we  shall  presently  show,  just  as 
concomitant  infections  with  verruga  and  malaria  commonly 
occur,  but  the  diseases  in  both  instances  are  entirely  independ- 
ent infections.  The  occurrence  of  malarial  attacks  in  indi- 
viduals during  the  stage  of  incubation  of  verruga  peruviana 
has  often  led  to  the  wrong  diagnosis  of  the  febrile  or  Oroya 
fever  stage  of  verruga  peruviana,  and  hence  to  a  confusion 
in  relation  to  the  symptomatology  and  other  clinical  features 
of  Oroj^a  fever  as  described  in  medical  literature.  Also  it 
seems  evident  from  the  published  accounts  that  cases  of 
infection  with  paratyphoid  fever  which  have  developed  verruga 
peruviana  have  been  confounded  with  Oroya  fever  or  Carrion's 
disease.  Demonstration  of  this  fact  is  given  by  the  investiga- 
tions of  several  observers.  Thus  Barton,  Biffi,  Carbajal, 
Tamayo,  and  Gastiaburu  have  isolated  paratyphoid  bacilli  ' 
from  the  blood  of  supi)osed  cases  of  Oroya  fever  or  of  Carrion's 
disease.  Obviously  it  is  possible  that  some  cases  of  Oroya 
fever  are  complicated  by  a  paratyphoid  infection.     It  seems 

'  Lor.  cit. 


18  OROYA  FEVER 

evident  that  it  is  largely  on  account  of  these  facts  that  the 
published  clinical  descriptions  of  uncomplicated  cases  of 
Oroya   fever   are   meager. 

From  the  study  of  the  literature  relating  apparently  to 
uncomplicated  cases  of  this  disease,  and  from  our  own  observa- 
tions upon  other  such  cases,  the  following  conclusions  have 
been  reached  in  regard  to  its  clinical  features. 

It  is  stated  that  the  incubation  period  is  about  20  days, 
and  our  observations  are  not  contrary  to  this  idea.  In  our 
opinion,  however,  this  statement  has  not  been  conclusively 
demonstrated,  and  we  were  unable  definitely  to  verify  it, 
owing  to  the  fact  that  we  did  not  have  an  opportunity  to 
observe  a  sufficiently  large  number  of  cases  from  which  to 
draw  definite  conclusions.  The  disease  is  usually  introduced 
by  malaise,  pains  and  weakness  in  the  limbs,  and  indisposition. 
Following  these  symptoms  slight  rigors  and  fever  appear. 
The  febrile  symptoms  and  rigors  usually  increase  in  severity 
as  the  disease  develops.  The  fever  is  often  very  irregular, 
being  usually  remittent,  but  sometimes  intermittent.  The 
temperature  frequently  fluctuates  between  100°  F.  and  102°  F., 
rarely  rising  to  104°  F.  The  fever  is  accompanied  by  a  rapid, 
pernicious  anaemia,  which  may  be  complicated  or  not  by 
hemorrhage,  vertigo,  or  syncope.  The  number  of  red  cells 
may  be  reduced  to  1,000,000  per  cubic  millimeter  or  less.  The 
skin  becomes  pale,  the  mucous  membranes  assume  a  waxy 
appearance,  and  murmurs  are  heard  over  the  heart  and  cervical 
vessels.  Oedema  of  the  legs  and  about  the  joints  may  occur. 
The  tongue  is  coated,  and  the  bowels  frequently  constipated 
in  the  early  stages.  Later,  diarrhea  frequently  develops. 
The  urine  is  scanty,  and  the  specific  gravity  high.  Albumin 
is  occasionally  present.  The  prostration  increases  as  the 
disease  progresses,  and  restlessness,  insomnia,  and  delirium 
may  develop.  In  uncomplicated  cases  there  is  no  eruption 
upon  the  skin.  It  has  been  assumed  by  some  writers  that  an 
eruption  in  these  cases  appears  in  the  internal  organs.  In 
such  an  opinion  we  do  not  concur,  and  the  results  obtained 
in  post  mortem  examinations  do  not  coincide  with  this  view. 


BLOOD  19 

In  general  it  may  be  stated  that  the  cases  of  Oroya  fever  are 
grave  ones  and  terminate  frequenth'  in  death.  Mild  cases 
which  terminate  favorably  do,  however,  occur,  and  we  had  an 
opportunit}^  to  stiid}^  infections  of  this  nature.  In  some 
instances  the  temperature  sinks  to  normal  or  subnormal  and 
death  occurs  from  excessive  exhaustion.  Death  may  occur 
within  a  few  weeks  of  the  onset  of  symptoms.  Some  observers 
report  that  death  maj-  occur  as  early  as  ten  days  from  the 
onset  of  the  attack,  but  we  have  not  observed  instances  of  such 
short  duration.  In  the  cases  which  recover,  improvement  in 
the  severe  symptoms  of  the  disease  usually  begins  within 
twenty-five  to  thirty  days,  and  convalescence  follows.  The 
mortality'  probably  equals  usually  from  30  to  40  per  cent. 
Some  observers  however  give  a  mortality  of  75  per  cent  and 
Rocha-Lima  states  it  ma}^  reach  98  per  cent.  The  spleen  is  fre- 
quently enlarged  but  according  to  the  observations  of  others  is 
not  always  palpable.  The  liver  may  be  moderately  swollen, 
and  the  lymphatic  glands  are  almost  always  more  or  less 
enlarged.  It  is  stated  that  pain  in  the  joints  is  a  common 
sj-mptom,  but  we  were  unable  to  verify  this  statement  in 
relation  to  uncomplicated  cases  of  the  disease.  Pain  in  the 
long  bones  is  sometimes  complained  of. 

Blood.  The  most  striking  and  characteristic  feature  in 
Oroya  fever  is  the  changes  which  are  found  in  the  blood. 
This  has  led  to  the  disease  being  sometimes  termed  in  Peru 
"  anaemia  perniciosa  de  los  quebradas."  The  development 
of  the  anaemia  in  severe  infections  may  be  very  rapid  and 
pernicious.  In  one  of  the  cases  studied  by  us,  which  will  be 
described  presently  in  detail,  the  red  blood-corpuscles  num- 
bered less  than  1,000,000  per  cubic  millimeter,  and  the  entire 
course  of  the  disease,  terminating  in  death,  was  but  twenty- 
eight  days.  Monge  '  reports  cases  in  which  the  number  of  red 
cells  fell  to  500,000  per  cubic  millimeter  in  a  very  short  period 
of  time,  and  death  followed  in  a  few  days.  Nucleated  red 
cells  appear  in  the  peripheral  circulation  early,  and  rai)idly 
increase  in  number.     Normoblasts  are  most  numerous,  liut 

'  Monge:  .1.  Lond.  School  Trop.  Med.,  1912,  i,  Part  3,  239. 


20  OROYA  FEVER 

megaloblasts  are  frequently  seen.  Polychromatophilia  and 
poikilocytosis  are  particularly  marked  in  severe  infections  in 
both  erythrocytes  and  erythroblasts.  Many  granular  cells 
are  visible.  Great  variation  in  the  size  of  the  erythrocytes 
also  occurs.  Monge  who  has  apparently  studied  uncompli- 
cated cases  of  Oroya  fever  reports  in  severe  ones  2,000  normo- 
blasts and  200  megaloblasts  per  cubic  millimeter.  In  one 
case  the  normoblasts  numbered  15,300,  and  the  megaloblasts 
3,420.  Leukocytosis  is  almost  always  present.  A  count  of 
20,000  is  common.  Differential  counts  often  show  nothing  par- 
ticularly characteristic  in  the  proportions  of  the  different  varie- 
ties. Bassett-Smith  ^  found  in  studying  the  blood  from  seven 
cases  that  the  polymorphonuclears  numbered  from  65  to  70 
per  cent,  the  large  mononuclears  from  7  to  10  per  cent,  the 
small  mononuclears  from  17  to  21  per  cent,  transitional  forms 
from  1  to  3  per  cent,  eosinophils  from  2  to  4.5  per  cent. 
Bifii  ^  found  the  polymorphonuclears  44  per  cent,  eosinophils 
10  per  cent,  large  mononuclears  16  per  cent,  lymphocytes 
30  per  cent.  In  the  severest  case  which  we  encountered  the 
polymorphonuclear  leukocytes  numbered  56  per  cent,  the  large 
mononuclears  16  per  cent,  transitional  forms  0.1  per  cent, 
small  mononuclears  12  per  cent,  eosinophils  0  per  cent :  Norm- 
oblasts 9  per  cent,  myelocytes  2.5  per  cent.  Myelocytes  are 
present  in  the  severe  cases  ranging  usually  from  0.5  to  2 
per  cent.  The  hemoglobin  in  severe  infections  may  amount 
to  but  15  per  cent.  In  less  severe  cases  of  infection  it  may  be 
from  40  to  50  per  cent.  In  severe  cases  the  parasites  which 
give  rise  to  the  disease  are  present  in  the  red  cells  in  great 
numbers,  but  in  mild  cases  the  number  of  infected  corpuscles 
is  small  and  a  long  search  is  often  necessary  to  disclose  the 
parasites.  The  following  notes  extracted  from  cases  observed 
by  us  will  serve  to  illustrate  special  clinical  features  of  the 
disease. 

1  Bassett-Smith:   Brit.  M.  J.,  Lond.,  1909,  ii,  783;   Lancet,  Lond.,  1909,  461;   Tr. 
Soc.  Trop.  Med.  and  Hyg.,  Lond.,  1914,  vii,  158. 

2  Biffi:  Bol.  Acad,  de  med.  de  Lima,  1903,  iii,  2. 


CLINICAL    CHART 


DATE  June  ib,  1S13. 


Day  of 

Month 


U27  2?2S30  I    2    5    Y    5    fc    7    8    «?  10  //  /2 
12  13   If  15  lb  17  l«  n  ZOll  22  23  2¥:i5  2t  27  22 


N' 


^Cascixz-    ffernicious    OTOva  FeveirL. 


ILLUSTRATIVE   CASES  21 

Cases  Emphasizing  Special  Clinical  Features 
Pernicious  Oroya  Fever 
Case  Id.  Pernicious  Oroya  Fever  terminating  in  Death.  —  The  patient, 
aged  twenty-three  years,  a  native  of  Austria,  was  received  in  the  service  of 
Dr.  Arce  in  the  Hospital  Dos  de  Mayo  of  Lima,  on  June  26,  1913.  We  are 
indebted  to  Dr.  Arce  for  notes  regarding  the  history  of  this  patient  and  for 
the  temperature  record.  The  patient  came  to  Peru  in  January,  1911. 
In  January,  1912,  he  suffered  with  malaria  and  anaemia.  In  October 
he  had  disturbance  of  the  bladder.  In  April  and  May  of  1913  he  traveled 
on  foot  through  the  quehrada  of  Matucana,  passing  the  night  at  San  Barto- 
lome,  which  is  known  to  be  one  of  the  most  dangerous  places  in  relation  to 
disease  in  the  verruga  zone.  In  the  middle  of  June  he  became  sick  with 
chills  and  fever,  which  attack  he  states  lasted  three  days.  Eight  days 
later  he  tried  to  return  to  his  work  but  had  to  abandon  it  on  account  of 
severe  fever  Avhich  was  accompanied  by  pains  in  the  body.  The  patient 
took  purgatives  on  several  occasions  and  this  was  followed  by  diarrhea 
and  tenesmus.  He  then  grew  rapidly  weaker.  The  fever  became  more 
intense  and  headache  and  delirium  developed.  He  Avas  not  able  to  walk 
or  stand,  and  was  brought  to  Chorillos  on  a  burro  and  from  there  to  the 
hospital  on  June  26.  Upon  entrance  the  patient  showed  marked  anaemia 
of  the  skin  and  mucous  membranes.  He  was  emaciated,  the  skin  was  dry, 
the  tongue  moist  and  furred.  The  lungs  appeared  normal.  There  was 
visible  pulsation  in  the  region  of  the  heart  and  the  large  vessels  of  the  neck. 
A  distinct  murmur  could  be  heard  in  the  pulmonary  area  over  the  heart. 
Gallop  rhythm  was  present.  Epigastric  pulsation  was  visible.  The 
inguinal  and  cervical  glands  were  swollen.  The  axillary  glands  were  just 
palpable.  The  liver  extended  just  below  the  costal  margin.  The  spleen 
was  just  palpable  and  firm  to  the  touch.  The  patient  complained  of  head- 
ache and  from  the  time  of  entrance  suffered  with  attacks  of  nausea,  vomit- 
ing and  diarrhea.  The  faeces  were  fluid  and  contained  bile.  Ova  of 
Trichocephalus  trichiurus  were  present  in  the  faeces.  The  analysis  of 
the  urine  showed  a  small  amount  of  albumin  and  urobilin  present.  The 
patient  gradually  sank,  a  systolic  murnmr  appeared  over  the  apex  of  the 
heart  and  diarrhea  increased.  A  moderate  oedema  of  the  legs  and  hands 
appeared. 

On  July  7  the  following  note  was  dictated  by  one  of  us: 

"  Patient  in  grave  condition,  temperature  100°.  7  F.  The  skin  presents 
most  extreme  grade  of  pallor.  There  is  very  marked  anaemia  of  the 
mucous  membranes  and  finger  tips.  Hemic  murmurs  are  heard  at  the 
apex  of  the  heart  and  over  the  cervical  vessels.  About  10  c.c.  of  blood 
were  drawn  from  the  vein  of  the  forearm  and  cultures  were  made  on  various 


22  OROYA  FEVER 

media.  Microscopical  preparations  were  also  made  of  blood  from  the  ear. 
Many  of  these  were  fixed  and  a  few  were  examined  as  fresh  preparations. 
Motile  parasites  were  observed  in  the  red  cells  in  the  fresh  preparations 
and  also  in  stained  ones.  The  Wassermann  reaction  is  negative  to  beef 
heart  antigen.  The  red  blood-corpuscles  measure  approximately  50,0,000 
to  the  cubic  millimeter.  It  is  difficult  to  count  the  red  blood-corpuscles 
on  account  of  the  fact  that  they  become  massed  together  in  rouleaux. 
The  white  blood  count  is  27,000  cells  to  the  cubic  millimeter,  without 
correction  for  the  normoblasts;  the  hemoglobin  15  per  cent  (Sahli);  the 
urine  shows  a  small  amount  of  albumin,  and  the  faeces,  ova  of  Tricho- 
cephalus  trichiurus.'' 

On  July  8  the  following  note  was  made : 

"  Specimens  of  fresh  blood  again  examined  from  8  to  11.30  a.m.  Defi- 
nite organisms  probably  allied  to  Anaplasma  were  observed  within  the 
red  cells.  These  were  very  numerous,  and  consisted  of  rounded  bodies  and 
also  of  elongated  rod-like  or  somewhat  spirillum-like  bodies.  The  rounded 
bodies  though  not  possessing  Brownian  movement,  nevertheless  moved 
about  in  the  red  cells,  and  frequently  changed  their  relative  position  to  one 
another.  In  some  cells  both  rods  and  rounded  bodies  could  be  observed. 
The  number  of  rounded  bodies  in  the  cells  frequently  equaled  three  or  four. 
The  rod-shaped  bodies  were  also  frequently  multiple.  They  possessed 
distinct  oscillatory  and  undulating  movements,  rotating,  and  freely  chang- 
ing their  position  within  the  blood-corpuscle.  The  rodrlike  bodies  were 
not  only  straight,  but  some  were  curved  and  some  were  almost  spirillum 
shaped.  In  one  corpuscle  which  was  watched  for  one  hour  and  fifteen 
minutes  there  were  two  rounded  bodies  and  one  rod-shaped  body.  The 
rod-shaped  one  possessed  distinct  oscillatory  movement,  changing  its 
position  freely  in  the  protoplasm  of  the  cell,  while  the  two  rounded  bodies 
also  changed  their  position  within  the  body  of  the  cell,  but  to  a  much 
less  extent.  At  times  the  rod-like  body  had  distinct  undulatory  move- 
ments, and  appeared  to  be  making  attempts  to  escape  from  the  periphery 
of  the  red  cell  as  it  moved  from  the  center  to  the  border  of  the  red  cell. 
During  one  period  of  the  observation  one  pole  showed  a  distinct  drum- 
stick-like appearance.  At  one  time  the  head  of  the  drumstick  seemed  to 
touch  one  of  the  rounded  bodies,  and  then  a  very  sudden  vibratory  motion 
occurred  throughout  the  rod-shaped  body,  the  extremity  lashing  back  and 
forth.  It  then  became  again  free.  About  half  an  hour  later  this  rod- 
shaped  body  seemed  to  have  become  much  shorter,  reaching  then  only 
about  half  its  fornier  length,  and  at  both  poles  there  appeared  to  be  a 
slightly  swollen  end.  The  rod-shaped  body  gradually  became  quieter, 
there  was  no  more  oscillation,  and  it  became  increasingly  difficult  to  see. 
At  times  it  appeared  as  though  it  was  more  in  the  depth  of  the  corpuscle 


ILLUSTRATIVE   CASES  23 

than  at  others.  It  was  watched  for  a  half-hour  further,  no  other  change 
occurred  in  the  cell,  except  that  the  bodies  became  very  difficult  to  dis- 
tinguish at  all.  The  rounded  bodies  seemed  to  consist  of  two  crescentic, 
more  opaque  bodies  almost  touching  one  another,  surrounded  by  a  more 
translucent,  rounded,  grayish  area." 

On  July  9  the  following  observation  was  made: 

"  The  fresh  blood  shows  apparently  a  smaller  number  of  the  rod-shaped 
and  rounded  bodies.  The  rounded  bodies  seem  to  consist  of  a  vacuole-like 
space  in  which  is  contained  a  small,  dot-sized,  less  translucent  area.  In 
stained  preparations  these  appear  as  a  small,  dot-sized,  compact  mass  of 
chromatin  contained  in  a  vacuole-like  space  evidently  representing  a 
protokaryon  of  the  smallest  type.  The  study  of  the  bacillary  bodies  today 
shows  that  when  the  slide  is  warmed  before  the  blood  specimen  is  taken, 
and  the  preparation  examined  immediately,  the  bacillary  forms  have  an 
oscillating  movement  within  the  red  corpuscle.  They  appear  to  be  made 
up  of  soft  protoplasmic  structure,  and  when  they  move,  the  limiting 
longitudinal  edges  of  the  organism  do  not  remain  as  straight  lines,  and 
the  organism  appears  at  these  times  to  have  a  greater  width  in  some 
places  than  in  others.  The  bacillary  forms  in  the  fresh  preparations 
frequent^  appear  bead-like  at  both  ends.  The  movement  of  the  bacillary 
bodies  is  entirely  unlike  that  of  pedesis,  but  is  an  oscillation  of  the  proto- 
plasm itself.  The  organism  sometimes  revolves  around  the  entire  circum- 
ference of  the  red  cell.  In  some  of  the  preparations,  which  were  taken 
upon  a  cold  slide,  the  movement  of  the  organisms  was  either  not  visible 
or  became  suspended  after  one  or  two  minutes." 

Numerous  parasites  were  also  found  in  the  stained  blood  specimens,  a 
detailed  description  of  which  will  be  found  on  p.  29  of  this  report.  On 
July  12  the  patient's  temperature  rose  to  102°. 7  F.  The  diarrhea  and 
vomiting  had  persisted.  The  tongue  was  dry  and  furred.  The  patient 
grew  weaker  and  weaker,  the  pulse  became  rapid  and  finally  could  no 
longer  be  felt,  and  he  died  at  2  p.m.  The  description  of  the  necropsy 
is  given  on  page  46. 

Concomitant  Infection 

Cases  of  verruga  peruviana,  Oroya  fever,  and  malaria  are 
frequently  found  in  the  same  endemic  regions,  and  we  have 
referred  to  the  fact  that  many  cases  of  verruga  peruviana  are 
complicated  by  malaria.  The  statistics  of  Peruvian  physicians 
demonstrate  conclusively  this  fact,  as  do  our  own  observations 
upon  cases  of  verruga  peruviana.     Sometimes  concomitant 


24  OROYA  FEVER 

infection  with  verruga  peruviana  and  Oroya  fever  occurs. 
The  following  case  observed  by  us  is  one  of  this  nature : 

Ca,se  20.  Mild  Case  of  Oroya  Fever  complicated  with  Verruga  Peruviana. 
Recovery.  —  Patient  was  first  seen  in  San  Bartolome  on  July  4.  At  this 
time  he  showed  a  marked  anaemia  and  complained  of  fever.  The  mucous 
membranes  showed  marked  pallor,  and  the  skin  was  distinctly  pale.  The 
physical  examination  of  the  chest  showed  nothing  abnormal  and  there  was 
no  visible  eruption  upon  the  skin.  There  were  many  cases  of  verruga 
peruviana  in  San  Bartolome  and  in  the  vicinity  of  his  dwelling  at  the  time. 
The  red  blood-corpuscles  numbered  3,300,000;  the  white  corpuscles 
4,500;  the  hemoglobin  50  per  cent  (Sahli);  the  specific  gravity  of  the 
urine  was  1.023,  but  nothing  abnormal  was  observed  in  it.  A  blood  smear 
was  made  from  the  case,  and  we  were  compelled  to  return  by  train  a  few 
minutes  later  to  Lima.  The  preparation  of  the  blood  was  examined  in 
lima,  and  found  to  contain  a  few  parasites  {Bartonella  bacilliformis) . 
On  July  23  the  patient  was  seen  again  for  a  few  minutes  and  blood  smears 
were  again  taken.  The  patient  still  showed  marked  anaemia,  pallor  of 
the  skin  and  of  the  mucous  membranes.  He  stated  that  he  was  growing 
weaker,  though  still  sitting  up  most  of  the  time.  On  the  legs  above  the 
ankles  four  or  five  reddish  papules  measuring  from  2  to  4  mm.  were  present. 
One  was  of  a  dark  cherry  color,  the  skin  over  it  appearing  tense  and  trans- 
lucent. There  was  also  one  lesion  measuring  3  mm.  in  diameter  on  the 
right  forearm.  These  evidently  represent  lesions  of  verruga  peruviana. 
On  returning  to  Lima  Bartonella  hacilliformis  was  found  in  small  numbers  in 
the  stained  preparations  of  the  blood.  The  patient  refused  to  come  to  the 
hospital  or  to  accept  medical  treatment,  and  would  only  allow  a  few  prepa- 
rations of  his  blood  to  be  taken  from  the  ear.  On  August  1  we  again 
visited  San  Bartolome  and  saw  the  patient  and  collected  microscopical 
preparations  of  the  blood.  No  parasites  were  found  in  these  blood  prepa- 
rations. 

The  following  note  was  made: 

"  The  eruption  of  verrugas  has  become  considerably  more  profuse  and 
distinct.  The  papules  are  now  situated  over  both  surfaces  of  the  right 
and  left  forearms.  They  measure  from  2  to  6  mm.  in  diameter,  are  red  in 
color,  and  raised  above  the  surface  of  the  skin.  They  are  quite  typical 
of  verruga,  and  there  are  about  thirty  of  the  larger  ones  on  both  forearms. 
The  palms  of  the  hands  and  soles  of  the  feet  are  clear.  There  are  also 
about  twenty  papules  on  the  forehead  and  left  side  of  the  cheek,  measuring 
2  to  3  mm.  in  diameter.  On  the  legs  there  is  a  similar  papular  eruption, 
particularly  on  the  anterior  surface  over  the  lower  portion  of  the  tibia. 
One  of  the  lesions  measures  1  cm.  in  diameter.  The  other??  are  small  in 
size.    The  patient  still  looks  pale  and  he  complains  of  much  pain  in  his 


ILLUSTRATIVE  CASES  25 

legs."  He  again  refused  to  enter  the  hospital  or  to  allow  any  of  the  lesions 
of  the  skin  to  be  removed  for  examination.  It  was  not  possible  to  see  this 
patient  again,  but  we  understood  that  he  eventually  recovered.  At  the 
time  he  suffered  from  Oroya  fever  there  were  in  his  immediate  neighbor- 
hood eight  cases  of  verruga  peruviana. 

]\IiLi)  Infection  with  Spurious  Eruption 

In  Peru  in  the  regions  where  Oroya  fever  and  verruga  peru- 
viana prevail  the  inhabitants  are  all  very  familiar  with  the 
disease  known  as  verruga,  and  with  the  inoculation  experiment 
of  Carrion.  It  is  the  popular  and  wide-spread  belief  in  Peru 
that  Carrion  inoculated  himself  with  blood  from  a  case  of 
verruga,  and  died  of  Oroya  fever,  subsequently  often  known  as 
"  Carrion's  disease."  The  inhabitants  in  these  regions  stand 
in  much  fear  of  the  febrile  stage  of  Carrion's  disease,  and  there 
is  a  general  and  implicit  belief  among  them  that  if  the  eruptive 
stage  of  the  disease  can  be  brought  on  by  treatment,  that  the 
individual  will  then  surcl}^  recover.  For  this  reason  when 
cases  of  severe  fever  and  anaemia  occur  various  remedies  are 
employed  by  the  natives  with  the  idea  of  causing  this  eruption 
to  develop.  In  this  way,  by  the  use  of  rubefacients  and  other 
substances  irritating  to  the  skin,  very  striking  cutaneous 
lesions  are  sometimes  produced.  If  the  individual  recovers 
from  the  attack  of  fever,  then  it  is  claimed  that  the  recovery 
from  the. fever  has  occurred  because  a  copious  and  abundant 
eruption  has  been  produced  upon  the  skin.  Obviously  many 
fevers  of  different  origin  are  treated  in  this  way.  Moreover 
every  papule  or  mole  or  naevus  upon  the  skin  overlooked  in 
the  previous  incomplete  examination,  but  subsequently  de- 
tected, is  pointed  out  enthusiastically,  frequently  even  by 
nurses  and  doctors,  as  an  evidence  of  the  appearance  of  the 
eruption.  Nothing  further  for  the  diagnosis  of  verruga  seems 
necessary  in  such  cases. 

The  following  case  illustrates  these  facts: 

Case  8.  Moderately  Severe  Oroya  Fever  termlnaling  in  Recovery.  —  The 
patient,  an  Italian  twenty-seven  years  of  age,  was  first  seen  on  July  4,  1913, 
in  San  Bartolome,  Peru,  a  well  known  endemic  center  of  Oroya  fever, 
verruga  peruviana,  and  malaria.     He  gave  a  history  of  having  Ijeen  in 


26  OROYA  FEVER 

San  Bartolome  for  seven  weeks  and  having  had  fever  for  three  weeks. 
He  was  employed  as  a  workman  upon  the  railway  until  he  became  sick. 
Physical  examination  of  the  chest  revealed  nothing  abnormal.  The 
spleen  was  not  definitely  palpable  and  the  liver  did  not  extend  below  the 
costal  margin.  The  temperature  at  the  time  was  101°. 3  F.,  the  pulse  one 
hundred.  The  mucous  membranes  Were  pale  in  color.  The  patient  was 
distinctly  anaemic.  A  thorough  examination  of  the  skin  showed  one  small 
papule  measuring  2  mm.  in  diameter  on  left  wrist,  grayish  in  color,  very 
slightly  raised,  and  rather  drj^  No  signs  of  an  inflammatory  process  in  it 
were  visible  and  it  apparently  had  little  significance.  On  the  skin  over  the 
anterior  surface  of  the  tibia  there  was  a  small  papule  measuring  about  3  mm. 
in  diameter,  and  slightly  raised.  It  was  gray  in  color  and  appeared  as  an 
old  dry  naevus.  No  other  lesions  were  visible  on  the  skin.  Fresh  and 
stained  microscopical  preparations  were  made  from  the  blood  and  20  c.c. 
were  withdrawn  from  the  median  basilic  vein  and  animals  inoculated  at 
once.  (These  inoculations  will  be  referred  to  elsewhere  in  the  report. 
See  page  36.)  The  red  blood-corpuscles  numbered  3,100,000;  the  white 
blood-corpuscles,  13,000;  the  hemoglobin  40  per  cent  (Sahh). 

The  differential  count  in  this  patient  was  not  as  characteristic  of  Oroya 
fever  as  Case  15,  partly  because  the  anaemia  did  not  progress  to  an 
extreme  grade  and  also  because  the  blood  picture  was  complicated  by 
extensive  application  to  the  skin  of  turpentine  and  other  rubefacients. 
This  treatment  was  probably  responsible  for  the  appearance  of  moderate 
amounts  of  albumin  in  the  urine  and  it  might  easily  have  modified  the 
blood  picture.     The  differential  count  is  as  follows: 

Polj^morphonuclear  neutrophils  72  % 

Large  mononuclears 10 

Lymphocytes 18 

Normoblasts 0.3 

Myelocytes none 

The  examination  of  both  fresh  and  stained  preparations  of  the  blood 
showed  a  few  parasites  of  Oroya  fever  (BoAonella  hacilliformis) .  The 
Wassermann  reaction  was  negative.  The  patient  refused  to  be  taken  to 
the  hospital  in  Lima  or  to  accept  medical  treatment,  saying  he  preferred 
to  be  treated  by  an  old  native  woman  who  had  had  experience  in  treating 
verruga  cases.  It  was  therefore  necessary  to  leave  him  in  San  Bartolome 
and  to  return  to  Lima.  The  patient  was  seen  again  on  July  23  at  San 
Bartolome.  During  the  interval  he  was  nursed  b}^  his  brother  who,  under 
the  direction  of  the  old  woman  referred  to  above,  carried  out  the  instruc- 
tions prescribed  by  her. 

The  following  note  was  made  on  this  date: 

"  His  brother  reports  that  he  has  been  much  improved  during  the  past 
week,  and  has  had  less  fever.     He  has  been  rubbing  him  with  leaves  of 


Fig.  1 

Parasite  of  Oroya  Fever  in  fresh  blood  specimen,  showing  changes 

in  shape  and  position  in  the  cell  that  the  organism  underwent 

during  a  period  of  IJ  hours  in  which  it  was  observed. 


Fig.  2 

Parasite  of  Oroya  Fever 

in  fresh  blood  specimen 

(bacillary  and  rounded  forms). 

Plate  IV  —  Oroya  Fever 


ILLUSTRATIVE  CASES  27 

several  kinds,  some  of  which  were  examined  in  a  fresh  state,  and  found  to 
be  those  of  the  castor  oil  plant  (Ricinus  communis).  Examination  of  the 
patient  shows  vesicular  dermatitis  over  the  face  and  legs,  where  the  leaves 
have  been  rubl^ed.  The  leaves  rubbed  on  evidently  have  had  powerful 
properties,  and  have  produced  lesions  resembling  strongly  those  due  to 
Rhus  venenata.  His  brother  states  that  these  are  verrugas  which  he  has 
caused  to  come  out  by  the  treatment.  There  is  no  resemblance,  to  an 
experienced  eye,  in  these  lesions  to  those  of  verruga  peruviana.  Blood 
smears  and  fresh  preparations  were  made  from  the  patient's  blood,  which 
were  examined  in  San  Bartolome,  and  also  at  the  next  station  a  half-hour 
later.  In  the  fresh  preparation  only  two  parasites  were  found,  one  of 
which  was  observed  for  an  hour  and  a  half.  This  observation  was  made 
at  Surco,  where  no  chair  or  table  was  obtainable,  and  it  was  necessary  to 
place  the  microscope  on  a  barrel,  and  stand  while  the  preparation  was 
studied.  In  the  field  of  the  microscope,  which  was  watched  for  an  hour 
and  a  half,  only  one  infected  corpuscle  was  visible.  The  other  corpuscles 
in  the  field  appeared  normal,  though  there  was  some  variation  in  size. 
There  was  but  one  polymorphonuclear  leukocyte  in  the  field  which  was 
not  pigmented.  The  parasite  was  situated  in  a  cell  which  appeared  normal 
in  other  respects.  It  was  bacillary  in  form,  and  had  an  undulating  move- 
.ment  similar  to  that  observed  in  the  rod  parasites  in  the  other  cases  of 
Oroya  fever.  At  times  it  was  difficult  to  see  the  parasite,  as  it  seemed  to 
be  deeper  in  the  corpuscle.  At  other  times  it  was  quite  sharp.  On  focus- 
ing, the  center  of  the  rod  seemed  more  dense  and  opaque  (darker),  and  as 
though  lying  in  a  vacuolated  space.  At  the  poles  sometimes  it  appeared  as 
though  a  dot  or  bead  were  visible,  but  this  was  not  distinct.  The  parasite 
moved  about  the  cell  slowly,  and,  as  may  be  seen  from  the  diagrams  which 
were  made  in  order  (Plate  iv.  Fig.  1),  it  in  turn  occupied  almost  every  part 
of  tfie  cell.  At  one  time  it  was  observed  just  at  the  peripherj" ;  at  another, 
it  was  perfectly  straight;  again  it  was  curved,  or  rather  sharply  bent 
in  the  middle;  at  other  times  bent  as  two  commas  joined  together,  never 
markedly  S-shaped,  and  never  pear-shaped,  but  occasionally  slightlj^ 
thicker  in  different  portions  of  the  body  when  moving.  The  movements 
were  slow,  and  never  suggested  Brownian  movement  in  any  way.  The 
undulations  gradually  became  slower,  but  otherwise  the  picture  did  not 
change  during  the  hour  and  a  half  of  observation,  when  it  became  necessary 
to  take  up  other  work." 

On  August  1  the  patient  was  again  seen.  The  following  note  was  then 
made : 

"  The  face,  hands,  forearms,  and  feet  and  legs  are  swollen  and  more  or 
less  puffy.  There  is  large  amount  of  dry  serum  on  the  surface  of  the  skin, 
and  numerous  scabs  which  have  formed  owing  to  the  l)reaking  of  follicles 
produced  In'  the  rubefacients  used  in  treatment.     Turpentine,  leaves  of 


28  OROYA  FEVER 

the  castor  oil  plant,  and  other  leaves  have  been  used  to  rub  the  skin. 
The  patient  has  also  been  given  various  concoctions  to  drink.  On  the  legs 
there  are  also  numerous  small  pin-point  and  pin-head  sized  hemorrhages, 
which  are  probably  the  result  of  irritation  and  scratching.  The  mouth 
and  lips  are  greatly  swollen  and  covered  with  sordes.  The  mouth  has  an 
appearance  as  though  the  patient  had  been  salivated.  The  tongue  is 
swollen,  and  there  are  several  small  fissures  at  the  edges.  On  the  mucous 
membranes  of  the  gums  and  the  palate  there  are  numerous,  closely  placed, 
small  (about  2  mm.  in  diameter)  nodular  areas.  On  the  glans  penis  there 
is  a  mass  measuring  about  1  cm.  in  diameter,  composed  of  similar  small 
nodules  which  are  all  confluent.  It  appears  not  unlike  a  soft  chancre. 
The  patient  says  he  has  no  fever,  but  refuses  to  allow  any  examination  to 
be  made.  Owing  to  the  treatment  he  has  received  it  is  impossible  to  form 
a  definite  conclusion  regarding  the  lesions  in  the  mouth  and  on  the  penis. 
All  the  lesions  present  on  the  skin  could  be  explained  as  the  result  of  the 
treatment."  We  were  also  denied  admittance  to  this  patient  subsequently 
but  were  informed  that  he  also  recovered. 

Description  and  Classification  of  the  Parasite  of 
Oroya  Fever  (Bartonella  bacilliformis) 

A  review  of  the  various  opinions  regarding  the  bodies  ob- 
served in  the  red  blood-cells  in  Oroya  fever  has  been  given  on 
pages  10-13. 

The  parasites  giving  rise  to  the  disease  were  observed  by  us 
in  the  peripheral  blood  of  Oroya  fever  cases  both  in  fresh  and 
in  stained  preparations.  The  organisms  consisted  of  minute 
rods  and  more  rarely  of  rounded  bodies,  situated  within  the 
red  cells.  Subsequently  other  stages  of  development  were 
discovered  in  the  endothelial  cells  of  blood-vessels,  particularly 
those  of  the  lymphatic  glands,  liver,  and  spleen. 

Fresh  Blood  Preparations. — ■  Although  in  fresh  blood  prepara- 
tions the  organisms  are  frequently  difficult  to  detect,  and  at 
times  lie  deep  in  the  substance  of  the  red  blood-cell,  neverthe- 
less with  good  illumination  and  an  oil-immersion  apochromatic 
objective,  at  times  they  may  be  distinctly  observed.  In  form 
they  are  rounded  or  rod-shaped,  though  the  rods  are  not 
always  straight  in  outline.  The  rods  measure  approximately 
from  1.5  to  2.5  m  in  length  and  the  rounded  bodies  from  0.5  to 
1  M  in  diameter.     In  severe  infections,  red  corpuscles  in  almost 


Fig.  1 


Fig.  2 


Plate  V  -Parasite  of  Oroya  Fever 

in  stained  blood  specimens. 


Figs.  1  and  2 

Parasite  of  Oroya  Fever 
in  stained  blood  specimen. 


Fig.  3 
Isolated  endothelial  cell. 


r^-ttr- 


Fig.  4 

Section  showing  cycle  of  Bartonella  bacilliformis 
in  endothelial  cells  of  lymphatic  glatid. 


Plate  VI  — Oroya  Fever 


DESCRIPTION   OF  PARASITE  29 

ever}^  field  of  the  microscope  are  invaded  by  the  parasites,  and 
numbers  of  both  rounded  bodies  and  bacillary  forms  are 
frequently  observed  in  a  single  cell.  The  organisms  are 
endowed  with  definite  motility,  which  amounts  to  slow  transi- 
tion and  is  totallj^  distinct  from  that  of  pedesis.  To  observe 
this  it  appears  advisable  previously  to  warm  the  slide  and  to 
examine  the  preparation  immediately  after  it  is  made.  In 
red  cells  in  which  several  of  the  parasites  are  visible  it  is  easy 
to  observe  their  frequent  change  of  position  within  the  cell 
and  with  reference  to  one  another.  The  rod-shaped  forms 
were  observed  to  glide  slowly  in  the  direction  of  their  long 
diameter  and  to  exhibit  a  slight  bending  in  their  transition  in 
the  red  cell,  and  at  times  both  these  and  the  rounded  bodies 
might  be  seen  to  occupy  in  turn  all  portions  of  it.  They  were 
never  observed  to  appear  as  distinct  cross  forms,  or  as  spirals 
or  markedly  S-shaped.  Occasionally  at  the  two  poles  of  the 
organism  a  dot  or  bead-like  appearance  was  observed.  On 
account  of  their  small  size  the  rounded  bodies  are  more  diffi- 
cult to  describe  in  fresh  specimens  and  it  can  only  be  stated 
that  they  change  their  position  within  the  cell.  A  more 
detailed  account  of  the  movements  of  the  parasite  is  given 
in  the  notes  on  pages  22,  23,  and  27.     See  also  Plate  iv. 

Stained  and  Fixed  Preparations.  —  The  rod-shaped  forms 
measure  approximately  from  1  to  2  /x  in  length  and  from  0.2  to 
0.5  IX  in  thickness.  They  are  frequently  slightly  curved,  and 
occur  singly  or  end  to  end  in  pairs,  or  in  chains  of  three,  four 
and  five.  When  numerous  they  often  lie  parallel  to  one 
another.  V  forms,  probably  representing  dividing  organisms, 
are  frequent.  Y  forms  are  also  not  uncommon.  Cross  forms 
are  rare  and  may  be  due  to  organisms  being  superimposed. 
The  ends  of  the  rods  in  stained  preparations  are  often  more 
intensely  colored.  Single  rods  sometimes  show  a  deep  red  or 
purplish  granule  which  may  be  of  the  nature  of  chromatin 
and  which  gives  the  appearance  of  a  swelling  at  one  extremity, 
the  rest  of  the  rod  having  a  more  bluish  tint,  sometimes 
slightly  deeper  at  the  end  opposite  the  one  showing  the  granule. 
Other  rods  may  be  blue  throughout  or  have  a  deeply  stained 


30  OROYA  FEVER 

granule  at  each  end.  In  organisms  occurring  in  chains  the 
deeply  staining  granules  sometimes  give  a  beaded  appearance 
to  the  chain  (Plate  v) .  It  is  evident  that  the  organisms  do  not 
all  lie  in  the  same  plane  within  the  corpuscle,  and  that  many 
of  them  are  not  superimposed  upon  it,  as  has  been  reported  by 
Lawson  ^  to  be  the  case  with  the  malarial  parasite.  This 
is  evident  from  some  of  the  photomicrographs. 

The  rounded  forms  measure  roughly  from  0.3  to  1  m  in 
diameter.  While  many  of  these  are  rounded,  others  are  slightly 
oval,  or  suggest  pear  shapes.  They  occur  singly,  in  pairs,  or  in 
groups  which  suggest  previous  division.     (Plates  v  and  vi.) 

The  red  cell  may  contain  a  variable  number  of  parasites. 
In  severe  infections  from  one  to  ten  is  not  unusual,  as  may  be 
seen  from  the  photomicrographs  (Plate  vii).  Nucleated  red 
cells,  at  various  stages  of  development,  are  of  frequent  occur- 
rence, and  are  sometimes  infected  with  the  parasites  (Plate 
VII,  Fig.  4) .  From  the  anaemia  which  occurs  in  this  disease 
and  the  number  of  red  cells  infected  with  parasites,  it  seems 
evident  that  the  red  cells  containing  the  parasites  are  ulti- 
mately destroyed. 

Classification 

From  the  description  given  above  of  the  parasite  as  ob- 
served in  the  red  blood-corpuscles  it  appeared  that  we  had  to 
do  with  a  species  of  organism  possessing  some  of  the  char- 
acteristics described  for  Anaplasma  or  of  Theileria,  but  also 
differing  in  other  characteristics  described  for  each  of  these 
genera.  The  rounded  bodies,  in  their  form  and  size,  and  in 
the  fact  that  they  apparently  consist  entirely  or  almost  entirely 
of  chromatinic  substance,  resemble  certain  bodies  which  were 
first  observed  by  Theobald  Smith  ^  and  later  by  Theiler  ^  in 
the  red  blood-corpuscles  of  cattle  afflicted  with  Texas  fever. 
Similar  forms  were  subsequently  found  in  the  red  blood- 
corpuscles  of  other  animals  afflicted  with  disease,  and  these 
bodies  are  now  classified  as  Anaplasmata.     The  bacilliform 

1  M.  Rowley-Lawson:  J.  Exper.  Med.,  1913,  xvii,  324. 

2  Smith:  Rep.  Bureau  Animal  Indust.,  Wash.,  1891-92,  177,  and  Plate  iv.  Figs. 
1,  2,  3,  p.  302. 

3  Theiler:  Ztschr.  f.  Infectionskrankh.  der  Haustiere,  1910,  viii,  39. 


^^ 


*"  V    , 


\ 


p%- 


Fig.  1.  —  Mild  infection.     Figs.  2,  :i,  4,  5,  6.  —  Severe  infection. 
Figs.  1,  2,  3,  4,  0.    =  X  1500.      Fig.  5.    =  X  2000. 


Plate  VII.  — Oroya  Fever  Parasite  in  Red  Blood-Cells 
(Stained  Phepahations). 


Fig.  1.  —  Lymph-xode.     Showing  swollen  endothelial  cells  in  blood-vessel  wall. 


Fig.  2.  —  Smkak  kkom  lvmimi-nodk.     I']m)()Tiii:mal  cklls. 
Showing  phase  in  iniilliplication  of  parasite.     Smear  staiiic<l  hy  (lieinsa'.s  method. 


Pl.ATK    VIII. OUOYA    FkVKU. 


CLASSIFICATION   OF   PARASITE  31 

or  rod-shaped  bodies  predominate  in  our  specimens,  and  in 
their  morphological  appearance  some  of  them  resemble  con- 
siderably the  rod-shaped  bodies  observed  in  certain  species  of 
Theileria.  Ho\Yever,  in  their  staining  reactions  they  are 
somewhat  different,  the  chromatinic  substance  is  not  differ- 
entiated from  the  cj^toplasmic  substance  with  the  same  ease, 
and  distinct  cross  forms  have  not  been  observed.  Moreover, 
their  movements,  so  far  as  could  be  observed,  were  unlike 
those  described  for  the  schizonts  in  the  Piroplasmata.  While 
the  organism  at  first  sight  might  be  regarded  from  its  mor- 
phology alone  as  a  species  of  bacterium,  this  idea  is  not  sup- 
ported by  further  study.  It  is  essentially  a  parasite  of  the 
red  blood-cell;  attempts  to  cultivate  it  on  various  culture 
media  have  been  unsuccessful  and  the  inoculation  of  large 
amounts  of  blood  from  severe  cases  of  Oroya  fever  has  failed 
to  infect  a  monke}^  or  rabbits. 

From  the  evidence  available  at  the  time  of  our  first  publica- 
tion on  the  subject  it  appeared  that  the  organism  observed  in 
the  blood  in  Oroya  fever  might  belong  to  a  group  of  micro- 
organisms intermediate  between  the  protozoa  and  the  bacteria, 
just  as  perhaps  the  Spirochaetae  form  another  such  group.  It 
resembled  in  some  of  its  characteristics  the  features  given  for 
two  species  of  Grahamella  N.  G.  Protista  described  and  classi- 
fied by  Brumpt  ^  in  October,  1911,  although  in  preparations 
stained  by  Giemsa's  or  Wright's  stains  the  presence  of  reddish- 
stained  granules  and  of  bluish  cytoplasm  in  many  forms 
favored  its  relationship  to  the  protozoa.  We  regarded  it 
preferable,  at  that  time  however,  until  further  information 
as  to  its  nature  was  acquired,  to  follow  Brumpt  in  his  classifi- 
cation of  Grahamella,  and  to  go  no  further  than  merely  to 
place  this  species  for  the  time  with  the  Protista.  From  the 
descriptions  in  the  literature  it  appeared  that  the  bodies 
described  by  Graham-Smith,^  Prowazek,^  and  others  in  the 

'  lirurnpt:   Bull.  Soc.  d.  pathol.  exotique,  Par.,  1911,  iv,  .514. 

2  Graham-Smith:  .J.  Hyg.,  Camb.,  1905,  v,  4.53;  Thomson:  ibid.,  19()r),  vi,  .574; 
Balfour:  Report  Wellcome  Trop.  Research  Lai).,  1906,  ii,  111;  1911,  iv,  120;  Bull.  See. 
d.  pathol.  exotique,  Par.,  1911,  iv,  060;  Leger:  ibid.,  1913,  vi,  247. 

'  Prowazek:  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  Jena,  1913,  Ixx,  Orig.,  34. 


32  OROYA  FEVER 

blood  of  moles  and  other  animals  and  classified  by  Brumpt  ^ 
and  Prowazek  ^  as  parasites,  might  constitute  organisms  of 
species  related  to  the  one  which  we  had  encountered  in  the 
blood  in  Oroya  fever.  As  in  the  genus  Grahamella  the  or- 
ganism of  Oroya  fever  is  characteristically  rod-shaped,  and 
evidently  multiplies  in  one  stage  by  binary  transverse  division. 
However,  the  resulting  elements  are  sometimes  observed  in 
chains.  On  the  basis  of  this  difference  and  of  other  additional 
characteristics  in  the  life  cycle,  motility,  staining  reactions 
and  size,  it  appeared  preferable  to  propose  for  this  organism 
the  name  of  Bartonia  hacilliformis.  The  generic  name  of 
Bartonia  was  suggested  owing  to  the  fact  that  Barton  in  1909 
inclined  to  the  belief  that  the  inclusions  earlier  observed  in 
the  red  cells  were  protozoa. 

Dr.  P.  W.  Bassett-Smith  has  since  kindly  called  the  atten- 
tion of  one  of  us  to  the  fact  that  the  name  Bartonia  had  been 
used  as  a  generic  one  in  botany  to  include  two  species  of  Gen- 
tianacese.  This  use  would  not  necessarily  preclude  its  employ- 
ment in  zoology  although  a  duplication  of  names  in  botany 
and  zoology  is  certainly  highly  undesirable.  Upon  investi- 
gation, however,  we  have  found  that  the  use  of  Bartonia  as 
a  generic  name  has  been  applied  by  Cossmann  ^  to  an  eocene 
moUusk,  Bartonia  canaliculatum,  hence  we  have  given  the 
generic  name  "  Bartonella,"  retaining  the  specific  one,  "  bacil- 
liformis  "  (Bartonella  hacilliformis). 

The  genus  was  also  defined  provisionally  from  the  forms 
studied  in  the  red  blood-corpuscles  as  follows:  organisms 
rounded  or  rod-shaped,  sometimes  occurring  in  chains  of 
several  dividing  forms;  reproduction  by  binary  transverse 
division;  cytoplasm  and  chromatinic  substance  often  differ- 
entiated with  difficulty;  endowed  with  independent  motility; 
living  as  parasites  in  the  red  blood-corpuscles. 

Development  within  the  Endothelial  Cells. —  Subsequently 
upon  examining  sections  of  the  organs  from  severe  fatal  cases 
of  Oroya  fever,  in  addition  to  the  pathological  changes  ob- 

^  Brumpt:  loc.  cit.  ^  Prowazek:  loc.  cit. 

^  Cossmann:  Essais  de  Paleoconchologie  Comparee,  4"^  Livr.,  Paris,  1901. 


O     X 


►J  tH 


a  — ' 

Eh      C 

z   2 


H     =3 


o 

o 


X 


'Z   _C: 


a  ii 


f^  .S 
o 


PARASITE   IX   ENDOTHELIAL   CELLS  33 

served  in  the  other  organs  and  described  on  page  49  of  this 
report,  other  evidences  of  the  hfe  cycle  of  the  parasite  were 
observed  in  the  swollen  endothelial  cells  of  the  lymphatic 
glands  and  spleen.  In  the  Ij^mphatic  glands  the  most  striking 
histological  feature  is  the  presence  of  large,  swollen,  endo- 
thelial cells,  sometimes  free  in  the  lumen  of  the  blood-vessel, 
at  other  times  still  attached  to  the  wall.  (Plate  viii.)  In  some 
instances  a  single  endothelial  cell  is  so  distended  that  it  will 
almost  occlude  the  entire  lumen  of  the  vessel.  These  cells 
are  distended  with  large  numbers  of  rounded  and  rod-shaped 
bodies  and  many  of  them  are  in  various  stages  of  degener- 
ation. Upon  the  study  of  smear  preparations  made  from 
the  lymphatic  glands  and  stained  by  Giemsa's  method, 
the  following  observations  were  made.  The  endothelial 
cells  in  these  preparations  contain  rounded  bodies  with  bluish 
cytoplasm  and  small  granules  of  chromatin  (Plate  x).  In 
some  of  these  bodies  but  a  single  granule  is  present;  in  others, 
two,  four,  or  many  may  be  observed.  Sometimes  the  cell  is 
distended  with  rounded  masses  or  spheres  each  of  which  con- 
tains a  definite  number  of  minute  points  of  chromatin.  (Plate 
X,  Figs.  2  and  3.)  These  spheres  are  of  quite  uniform  size. 
From  a  study  of  other  cells  it  would  appear  that  these  spheres 
break  up  into  a  large  number  of  minute  elements  each  of 
which  contains  a  chromatin  granule;  these  become  elon- 
gated and  finally  appear  as  distinct  rods  containing  at  one 
end  a  minute  particle  of  chromatin.  (Plate  x.  Fig.  5.) 
This  latter  appearance  is  particularly  apparent  when  the 
sphere  is  ruptured  and  the  rods  set  free.  If  these  rod- 
shaped  elements,  which  have  been  set  free  by  the  rupture  of 
the  endothelial  cells,  are  compared  with  the  rods  found  within 
red  blood-corpuscles  lying  near,  it  is  seen  that  they  are  identical 
in  character.  Moreover  they  are  entirely  similar  to  the  forms 
already  observed  in  the  blood-cells  in  the  peripheral  circula- 
tion. It  seems  very  probable  that  the  rods  which  have 
escaped  from  the  spheres  constitute  gametocytes,  rather 
than  merozoites,  which  have  subsequently  entered  the  ery- 
throcytes, and  the  movements  of  the  parasites  observed  in 


34  OROYA  FEVER 

the  red  blood-corpuscles  in  fresh  preparations,  and  already 
referred  to  in  this  report,  coincide  with  this  idea.  Thus  the 
appearances  observed  in  the  endothelial  cells  of  the  lymphatic 
glands  from  Oroya  fever  cases  resemble  considerably  those 
described  and  pictured  by  Gonder  ^  in  his  description  of 
the  life  cycle  of  Lympho-haematocytozoon  parvum  (formerly 
Pirosoma  parvum),  (Plate  xi,  Fig.  1).  Through  the  kindness  of 
Dr.  K.  F.  Meyer  one  of  us  recently  received  microscopical 
preparations  of  this  organism,  and  from  a  study  of  these 
preparations  and  the  articles  by  Meyer  ^  and  Gonder  upon 
this  subject,  the  similarity  between  the  organism  observed 
by  us  in  the  endothelial  cells  in  Oroya  fever  and  Lympho- 
haematocytozoon  parvum  is  further  suggested.  Owing  to  the 
more  minute  size  of  Bartonella  hacilliformis,  however,  particu- 
larly in  the  multiplicative  phase,  its  life  cycle  within  the  endo- 
thelial cells  is  much  more  difficult  to  observe  and  interpret. 
From  these  observations,  it  appears  that  Bartonella  hacilli- 
formis should  be  classified  as  a  protozoan  and  that  it  is  related 
to  the  Lympho-haematocytozoa,  a  genus  recently  created 
by  Meyer.  However  further  observations  upon  a  larger 
amount  of  material  are  desirable. 

Attempts  to  Infect  Animals  with  the  Parasite  of 
Oroya  Fever  {Bartonella  hacilliformis) 

Numerous  attempts  were  made  to  infect  animals  with  the 
parasite  observed  in  the  blood  of  Oroya  fever  cases.  These 
experiments  are  of  particular  interest  and  importance  in 
relation  to  the  differentiation  of  Oroya  fever  from  verruga 
peruviana,  and  form  one  link  in  the  chain  of  evidence  that  the 
two  diseases  are  distinct  and  have  an  entirely  different  etiology. 
Our  experiments  show  that  we  were  able  with  little  difficulty 
to  infect  animals  with  the  virus  of  verruga  peruviana,  but  our 
attempts  to  inoculate  animals  with  Bartonella  hacilliformis 
were  unsuccessful.  The  experiments  undertaken  to  inoculate 
animals  with  this  haematozoon  will  first  be  outlined. 

1  Gonder:  J.  Comp.  Path.  &  Therap.,  Edinb.  &  Lond.,  1910,  xxiii,  328. 

2  Meyer:  Kolle  &  Wassermann,  Handb.  d.  Path.  Mikroorganismen,  Jena,  2. 
Auflage,  1913,  vii,  531,  539. 


if  ^ 


^v 


£»'• 


Plate  X  — Oroya  Fever 

Endothelial  Cells 

showing  various  stages  in  the  development  of  the  parasite; 

early  rounded  forms  later  breaking  up  into  rods. 


;.  «•. 


m 


m 


m 


c' 


:# 


%;; 


'  "^'.-ii-.- 


^  •".  '(' 


1 


Fig.  1.  —  Life  cycle  of  Lympho-haematocytozoon  parvum. 


Fig.  2.  —  Ulceration  in  large  intestine. 


Plate  XI.  —  Oroya  Fever. 


ANIMAL   EXPERIMENTS  35 

Testicular  inoculations  in  rabbits  were  first  made  with  the 
idea  of  detecting  or  of  excluding  spirochaetal  infection. 
Testicular  inoculations  with  the  virus  of  verruga  peruviana 
produce  definite  lesions  but  not  of  spirochaetal  origin. 

Experiments 

I.  July  7,  1913.  10  c.c.  of  blood  were  drawn  from  the  median  basilic  vein  of  a 
severe  case  of  Oroya  fever  (Case  15)  which  resulted  fatally  five  daj^s  later.  The 
microscopical  examination  of  the  blood  on  this  date  revealed  a  very  severe  infection 
with  Bartonella  hacilliformis.  Animals  were  inoculated  with  the  blood  at  this  time 
and  the  notes  of  the  experiments  are  as  follows : 

Rabbit  No.  33.  —  Testes  inoculation  with  Oroya  blood. 

July  7,  1913.     Both  testes  (small)  inoculated  with  0.5  c.c.  of  defibrinated  blood 
of  case  at  Dos  de  INIayo  Hospital  about  1^  hours  after  removal. 
July    9.     11.45  a.m.     Temp,  ior.6  F.    Testes  small,  normal. 

"      11.     12.15    "  "       102°.3.  "'  "         firm,  normal 

"      15.     11.35    " 

"      18.     12       M.    '         "       102°.  "  "  "  " 

«      21.     11.40  a.m.  "       102°.5. 

Aug.    3. 

Rabbit  No.  34-  —  Testes  inoculation  with  Oroya  blood. 

Juh'  7,  1913.     Both  testes  each  inoculated  with  0.5  c.c.  of  defibrinated  blood 
of  Case  15  at  Dos  de  Mayo  Hospital  about  1§  hours  after  removal. 
July  9.     Rabbit  killed  and  partially  eaten  by  dogs. 

Rabbit  No.  35.  —  Testis  inoculation  with  Oroya  blood. 

July  7,  1913.  Right  testis  only  inoculated  with  0.5  c.c.  of  defibrinated  blood 
about  1^  hours  after  removal  from  case  at  Dos  de  Mayo  Hospital. 

Right  testis  and   scrotum  indurated 

and  considerable  oedema  in  scrotum. 

Left  not  found. 

Right  testis  normal.     Left  not  found. 


July     9. 

11.50  a.  m. 

Temp. 

lOr.GF. 

"      11. 

12.15    " 

u 

101°.9. 

«      15. 

11.37    " 

«      18. 

12.02  P.M. 

u 

102°.l. 

«      21. 

11.44  a.m. 

u 

102°.3. 

Aug.    3. 

Rabbit  No.  36.  —  Intravenous  inoculation. 

July  7,  1913.  One  cubic  centimeter  of  defibrinated  blood  from  case  at  Dos  de 
Mayo  Hospital  injected  into  ear  vein  about  1§  hours  after  removal. 

July    9.     11.45  a.m.     Temp.  99°.6  F.    Tail  is  soiled  with  faecal  material.     No 

parasites  visible  in  the  blood. 
"      11.     12.15    "  "     102°.4.       No  parasites  visible  in  the  blood. 

«      15.     11.35    "  "  "  "        "      " 

«      18.     12.03  P.M.  "     ior.5.         "  "  "        "      " 

«     21.     11.46  a.m.  "     102°.2.         "  "  "        "      " 

August  8.  Killed.  A  male  with  small  testes.  Organs  appear  normal.  No 
tissue  saved.  Blood  smear  made.  No  parasites  were  observed  in  the  blood  of 
the  animal  the  day  following  the  inoculation  or  subsequently. 


36 


OROYA  FEVER 


Monkey  No.  1 .  —  Intravenous  inoculation. 

July  7,  1913.  Two  cubic  centimeters  of  defibrinated  blood  from  case  at  Dos  de 
Mayo  Hospital  injected  intravenously  in  vein  of  leg. 

r  Blood  Smears  ) 
July    9.    11.55  a.m.   Temp.  101°  F.  Slight  diarrhea  j      Wright  and  [- Negative. 

(     Giemsa         ) 
«      11.    12.25  p.M.^       "      102°. 
«      13. 

"  15.  12.30  "  "  100°.9. 
"  17.  10.30  a.m.  "  101°.8. 
"      18.    12.25  P.M.        "      101°.9. 

II.  July  6,  1913.  lOc.c.  of  blood  were  drawn  from  vein  of  the  arm  of  a  rather 
severe  case  of  Oroya  fever  (Case  8)  in  which  the  parasites  (Bartonella  hacilliformis) 
were  demonstrable  by  microscopic  examination  in  fair  numbers.  Animals  were 
immediately  inoculated  with  this  blood  and  the  protocols  of  the  experiments  are 
as  follows: 

Rabbit  No.  20.  —  Testes  inoculation  with  Oroya  blood. 

July  6,  1913.  Both  testes  inoculated  with  0.5  c.c.  of  defibrinated  blood  from 
Italian  at  San  Bartolome  within  six  minutes  after  removal. 

July    9.     11.15  a.m.     Temp.  101°.6  F.    Both  testes  normal. 
"      11.     11.45    " 

«      15.     10.55    "  "       101°.8. 

«      18.     10.43    "  "       101°.7. 

"     21.     11.13    "  "       101°.6. 

"     26.     12.13  p.m. 
July  28.     Killed  by  dogs.     Hemorrhage  in  nostrils,  peritoneal  cavity,   and 
bitten  in  various  parts  of  the  body  through  the  skin.     Both  testes  appear  normal. 
Organs  present  no  lesions  apart  from  those  produced  by  biting.     No  tissue  saved. 

Rabbit  No.  21 .  —  Testes  inoculation  with  Oroya  blood. 

July  6,  1913.  Both  testes  inoculated  with  0.5  c.c.  of  defibrinated  blood  from 
Italian  at  San  Bartolome  within  eight  minutes  after  removal. 


July    9. 

11.15  a.m. 

Temp. 

101°.5  F. 

Both  testes  firm. 

"      11. 

11.45    « 

Right  testis  normal;  left  testis  homo 
geneous. 

"      15. 

11. 

u 

102°.4. 

Both  testes  firm,  normal. 

"      18. 

11.40    " 

a 

101°. 

a               u               a                11 

"     21. 

11.16    " 

a 

102°.l. 

a               u               11               u 

"     26. 

12.13  p.m. 

u              u               u                u 

«     30. 

a              u              u               u 

Rabbit  No.  22.  —  Testes  inoculation  with  Oroya  blood. 

July  6,  1913.     Both  testes  inoculated  with  0.5  c.c.  of  defibrinated  blood  from 
Italian  at  San  Bartolome  within  ten  minutes  after  removal. 
July    9.     11.20  a.m.     Temp.  101°.8  F.    Both  testes  normal. 


11. 

11.50 

(( 

Testes  small,  firm  and  normal. 

15. 

11.03 

u 

Testes  smaU,  firm  and  normal. 

18. 

11.45 

li 

«       101°.5. 

Testes  normal. 

21. 

11.18 

u 

«       102°.  1. 

U                           <( 

26. 

((                   » 

30. 

a                  a 

BLOOD   CULTURES  37 

Rabbit  No.  23.  —  Intravenous  inoculation  wdth  Oroj'a  blood. 
July  6,  1913.     2  c.c.  of  defibrinated  blood  from  Italian  at  San  Bartolome  in- 
jected into  right  ear  vein. 

July    9.    11.20  a.m.     Temp.  100°. 6  F.    Blood  smear  (Giemsa)  negative  for  para- 
sites. 
"      11.    11.50    "  "      lOr.6.       Rabbit  appears  normal.     Blood  smear 

(Giemsa)  negative  for  parasites. 
"      15.    11.05    "  "      102°. 3.       Rabbit  appears  normal.      Blood  smear 

negative. 
"      IS.    11.4:7    "  "      100°.5.       Blood  smear  (Giemsa)  negative. 

"     21.    11.20    "  "      102°.3. 

July  29.  Dead.  Yesterday  noted  ear  which  was  perforated  as  though  by  a  bite. 
At  the  autopsy  the  bladder  was  distended,  the  kidneys  showed  depressed  red  areas 
on  the  surface  1  mm.  in  diameter.  Other  organs  normal.  Skinned,  and  brain 
examined.  No  evidence  of  violence.  Tissue  in  Zenker's  solution.  Smear  stained 
by  Wright's  method;  no  parasites  in  the  blood. 

Conclusions. —  Thus  it  will  be  seen  from  these  experiments 
that  we  were  not  successful  in  infecting  animals  with  Bar- 
tonella hacilliformis,  either  by  intravenous  or  intratesticular 
inoculation.  Intravenous  inoculation  of  a  monkey  and  of 
rabbits  with  large  amounts  of  defibrinated  blood  from  severe 
cases  of  Oroya  fever  did  not  produce  any  noticeable  results, 
and  the  parasites  observed  in  the  blood  in  the  cases  in  man  were 
not  found  in  the  blood  of  the  inoculated  animal.  Obviously 
if  further  cases  for  study  had  been  at  our  disposal,  experiments 
of  this  nature  would  have  been  continued. 

On  the  other  hand,  we  were  able  to  transmit  successfully 
the  virus  of  verruga  peruviana  to  rabbits,  monkeys,  and  dogs, 
and  to  produce  definite  lesions  in  these  animals.  An  account 
of  these  experiments  is  given  in  Section  iv,  pages  82-125. 

Experiments  in  the  Cultivation  of  Microorganisms 
FROM  Blood  of  Oroya  Fever  Cases 

Attempts  were  also  made  to  cultivate  Bartonella  hacilli- 
formis in  vitro,  and  to  ascertain  as  well  if  bacteria  were  present 
in  the  blood  in  uncomplicated  cases  of  Oroya  fever.  On  July  6, 
10  c.c.  of  blood  were  drawn  from  an  arm  vein  of  a  case  of  Oroya 
fever  (Case  8),  in  whose  blood  at  the  time  fair  numbers  of  red 
blood-corpuscles  infected  with  Bartonella  hacilliformis  were 
demonstrated  by  microscopical  examination.  Cultures  were 
immediately  made  with  portions  of  this  blood  upon  the  com- 


38  OROYA  FEVER 

mon  bacteriological  laboratory  media  and  by  Noguchi's 
method  and  with  the  media  of  Bass.  The  inoculation  of  the 
blood  upon  the  culture  media  was  made  as  follows: 

3  tubes  of  ascitic  fluid  covered  with  paraffin  oil. 

1  tube  of  ascitic  fluid  (no  covering  of  oil). 

2  tubes  of  ascitic  fluid  agar  +  rabbit's  testicle. 
2       "       "  blood  serum. 

2  "  "  alkaline  agar. 

2  "  "  blood  agar,     (n  N  N  medium.) 

2  "  "  bile. 

4  "  "  Bass'  media. 
2  "  "  bouillon. 

All  of  these  cultures  with  the  exception  of  one  of  the  bouillon 
tubes  remained  apparently  sterile.  This  culture  was  con- 
taminated by  a  spore-bearing  bacillus.  In  the  other  tubes  of 
media,  during  the  ten  days  following  their  inoculation,  micro- 
scopical examination  and  the  gross  appearances  of  the  cultures 
did  not  reveal  or  suggest  any  growth. 

On  July  7  about  10  c.c.  of  blood  were  drawn  from  a  vein  of 
the  forearm  of  a  severe  case  of  Oroya  fever  (Case  15),  in  whose 
blood  large  numbers  of  parasites  (Bartonella  hacilliformis) 
were  present  at  the  time.  A  number  of  cultures  were  made 
with  portions  of  the  blood  as  follows : 

2  tubes  ascitic  fluid  (much  blood  inoculated)  covered  with  paraffin  oil. 

2  "          "           "      (much  blood  inoculated)  not  covered  with  paraffin  oil. 

4  "          "           "     agar  +  rabbit's  testicle. 

2  "          "           "     +  rabbit's  testicle. 

2  "  bile  (large  amount  of  blood  inoculated). 

2  "  blood  agar,     (n  n  n  medium.) 

3  "  Bass'  media. 

2  flasks  bouillon  (large  amount  of  blood  inoculated). 

All  of  these  cultures  remained  sterile  except  as  follows.  In 
one  tube  of  blood  agar  a  growth  developed  which  proved  to 
be  that  of  a  large,  thick  bacillus  apparently  a  saprophyte. 
One  of  the  tubes  containing  ascitic  fluid  and  blood  became 
distinctly  cloudy,  but  microscopical  examination  did  not 
reveal  the  presence  of  any  organism.  In  some  of  the  cultures 
the  red  blood-corpuscles  were  still  fairly  well  preserved  after 
forty-eight  hours,  but  no  parasites  could  be  distinguished 
longer  in  them. 


BLOOD   CULTURES  39 

On  July  11  blood  cultures  were  made  again  from  this  same 
case  the  blood  being  collected  less  than  twenty-four  hours 
before  death,  and  inoculated  upon  four  tubes  of  Bass'  media, 
three  of  blood  agar,  two  flasks  of  bouillon,  and  two  tubes  of 
bile.  AU  of  these  cultures  remained  sterile.  No  distinct 
development  of  the  parasites  of  the  red  cells  was  observed  in 
the  tubes  containing  blood  agar  or  Bass'  media.  Cultures 
were  also  made  from  this  case  at  the  autopsy  which  occurred 
the  following  day.  The  culture  from  the  heart's  blood  showed 
after  twenty-four  hours  several  large  white  colonies,  and 
microscopical  examination  revealed  a  large  spore-bearing 
bacillus.  The  organism  was  not  pathogenic  by  intraperitoneal 
inoculation  into  two  guinea  pigs,  and  evidently  represented  a 
contamination  from  the  air  or  an  infection  entering  just  prior 
to  death.  The  cultures  from  the  spleen  of  this  case  showed 
neither  typhoid  nor  paratyphoid  bacilli. 

Unfortunately  we  did  not  have  other  favorable  opportuni- 
ties for  attempting  the  cultivation  of  the  organisms  of  the 
red  blood-cells;  nevertheless  the  bacteriological  blood  examina- 
tions of  these  cases  of  Oroya  fever  are  important  in  connection 
with  the  previous  studies  of  several  investigators  in  relation  to 
Carrion's  fever  and  verruga  peruviana.  In  considering  the 
previous  investigations  which  were  carried  on  in  regard  to  the 
etiology  of  Oroya  fever  and  verruga  peruviana  in  Section  ii 
attention  was  called  to  the  fact  that  Barton,  Bifii,  Carbajal, 
Tamayo,  and  Gastiaburu  have  repeatedly  isolated  para- 
typhoid bacilli  from  the  blood  of  such  cases.  Tamayo  ^  in 
one  of  his  most  recent  pubhcations,  analyzed  thirty  cases  of 
verruga  peruviana.  In  twenty  of  these  paratyphoid  bacilli 
were  isolated  from  the  blood.  The  organisms  were  not  care- 
fully identified,  and  although  five  necropsies  were  performed, 
it  is  stated  that  in  three  nothing  special  was  found,  and  that 
one  was  negative.  In  the  fifth  the  remark  is  made  that  there 
were  a  few  verrugas  on  the  spleen.  However,  from  these 
bacteriological  examinations,  and  from  those  of  other  obser- 

'  Tamayo:  Cr6n.  m6d.,  Lima,  1905,  xxii,  No.  406,  p.  33.5;  No.  407,  p.  349;  J.  Trop. 
Med.,  Lond.,  1906,  ix,  1.59. 


40  OROYA  FEVER 

vers,  particularly  of  Biffi,  it  appears  that  paratyphoid  infection 
occurs  not  uncommonly  in  some  of  these  narrow  valleys  in 
Peru.  It  has  been  suggested  before  that  Carrion  died  of 
paratyphoid  fever  and  it  would  seem  that  one  might  be  justi- 
fied in  the  idea  that  Carrion's  disease  is  really  a  form  of  para- 
typhoid fever,  or  at  least  has  been  considered  as  such.  It  is 
interesting  to  note  that  Eder  ^  concludes  that  Carrion's 
pernicious  fever  is  typhoid  occurring  in  the  tropics,  but  that 
he  bases  his  conclusions  particularly  on  Tamayo's  observations. 
On  the  other  hand  it  is  obvious  that  paratyphoid  infection 
does  not  give  rise  to  the  rapid,  pernicious  anaemia  observed 
in  Oroya  fever  cases.  This  fact  alone,  from  a  clinical  stand- 
point, refutes  at  once  the  argument  that  Oroya  fever  is  a  form 
of  paratyphoid  infection.  Moreover,  paratyphoid  bacilli 
have  not  been  found  in  our  cases  of  uncomplicated  Oroya 
fever  infection,  and  even  in  Tamayo's  cases  such  organisms 
were  not  isolated  from  all.^  The  most  important  fact,  how- 
ever, is  that  in  Oroya  fever  a  specific  parasite  is  present  in  the 
red  blood-corpuscles  which  obviously  is  not  encountered  in 
uncomplicated  paratyphoid  infections.  The  fact  that  Biffi 
and  Carbajal  ^  and  Tamayo  ^  claimed  the  isolation  of  para- 
typhoid bacilli  from  suppurating  granulomata  in  cases  of 
verruga  peruviana,  further  complicates  the  question.  As 
Darling  ^  tersely  remarks,  "  to  those  who  have  had  any 
experience  with  infections  by  members  of  the  typhoid-colon 
group  in  the  tropics,  and  in  view  of  our  knowledge  of  the 
history  of  the  relation  of  Bacillus  icteriodes  and  Bacillus  x  of 
Sternberg  to  yellow  fever,  and  the  relation  of  the  hog  cholera 
bacillus  to  true  hog  cholera,  which  as  DeSchweinitz  and  Dorset 
have  shown,  is  due  to  a  filterable  virus,  these  bacteriologic 

1  Eder:  J.  Trop.  Med.,  Lond.,  1906,  ix,  213. 

2  In  our  Case  15,  dying  from  Oroya  fever,  no  ulceration  or  hyperplasia  of  Peyer's 
patches  was  found  in  the  intestine,  but  this  fact  would  not  necessarily  exclude  paraty- 
phoid infection  alone.  In  1902  one  of  us  (Strong,  Johns  Hopkins  Hosp.  Bull.,  Bait., 
1902,  xiii,  107)  reported  the  first  fatal  case  of  paratyphoid  infection  with  necropsy  with  the 
isolation  of  the  organism  from  the  spleen,  and  in  this  case  there  were  no  intestinal  lesions. 

^  Biffi  and  Carbajal:  Cron.  med.,  Lima,  1904,  xxi,  No.  379,  p.  285. 

*  Tamayo:  ibid.,  1905,  xxii.  No.  407,  p.  352. 

5  Darling:  J.  Am.  M.  Ass.,  Chicago,  1911,  Ivii,  2072. 


EXPERIMENTS   WITH   MOSQUITOES  41 

findings  of  Barton  and  Biffi  are  verj^  interesting,  and  thej^  add 
another  disease  to  the  Ust  of  those  frequently  compHcated  b^^ 
infections  with  bacilli  of  this  group." 

Obviously,  also,  in  the  isolation  of  the  organisms  in  Lima, 
from  so  many  of  these  cases,  the  technique  of  isolation  must  be 
considered  as  well  as  the  methods  employed  in  identifying 
the  organisms  encountered. 

From  this  discussion,  however,  and  that  given  elsewhere  in 
Section  iii,  it  is  evident  that  Oroya  fever  unquestionably  has 
been  confused  with,  or  wrongly  diagnosed  as  a  form  of  para- 
typhoid infection,  as  a  stage  of  verruga  peruviana  infection, 
and  as  a  form  of  malarial  fever. 

Attempts  to  Infect  and  Observe  Development  of 
Bartonella  Bacilliformis  in  Mosquitoes 

On  July  10  and  11  a  large  number  of  mosquitoes  were 
allowed  to  bite  a  severe  case  of  Oroya  fever  (Case  15),  in  the 
blood  of  which  at  the  time  many  red  blood-corpuscles  were 
infected  with  Bartonella  hacilliformis.  The  mosquitoes  used 
in  these  experiments  were  collected  as  larvae  from  a  concrete 
water  tank  at  Matucana,  a  well  known  endemic  center  of 
Oroya  fever.  The  larvae  were  transferred  to  Lima  and  adults 
were  reared  from  them  in  the  laboratory.  The  adults  were 
placed  in  small  cans  containing  sod  with  growing  grass  and 
covered  with  netting.  They  were  allowed  to  bite  through 
the  net  and  also  directly  when  removed  from  the  cans  and 
placed  in  test  tubes.  No  bites  were  secured  at  first,  but  aftef 
a  few  days  the  insects  evinced  a  greater  desire  to  feed.  None, 
however  appeared  to  be  as  hungry  as  is  usually  the  case  with 
other  species  of  mosquitoes,  and  none  became  fully  engorged. 
Several  of  these  mosquitoes  which  had  imbibed  blood  were 
dissected  and  examined  microscopically  daily,  the  first  exami- 
nations being  made  48  hours  after  the  feeding.  The  examina- 
tions were  continued  up  to  July  18  when  all  the  mosquitoes 
had  been  killed  and  examined.  No  evidence  of  the  presence 
of  the  parasites  was  discovered  in  the  stomach  or  salivary 
glands  and  no  oocytes  were  observed.     On  account  of  the 


42  OROYA  FEVER 

minuteness  of  the  organism  in  question,  the  negative  findings 
in  this  instance  should  not  be  regarded  as  final  or  conclusive. 
Unfortunately  we  were  unable  to  pursue  these  experiments 
further  on  account  of  not  being  able  to  obtain  suitable  material. 
The  mosquitoes  pi^oved  later  to  represent  an  undescribed  genus 
and  have  been  named  Phalangomyia  dehilis  by  Dyar  and  Knab. 
(Plate  XXXIII.)  The  genus  is  closely  related  to  Culex  as  is  shown 
by  both  adult  and  larval  characters.  No  further  specimens 
were  taken,  so  that  it  is  impossible  to  state  whether  the  species 
is  generally  distributed  through  the  Oroya  fever  zone,  or  pecu- 
liar to  it.  It  would  seem,  however,  that  both  assumptions  are 
not  unlikely.  It  appears  very  probable  that  Oroya  fever  is 
transmitted  by  the  bite  of  some  arthropod,  and  the  demon- 
stration of  whether  this  fact  is  true  should  not  be  difficult.  The 
fact  that  the  parasite  is  present  in  such  large  numbers  in  the 
peripheral  circulation  naturally  suggests  that  it  is  transmitted 
by  some  blood-sucking  insect  which  serves  as  an  intermediate 
host.  The  failure  to  infect  either  the  monkey  or  the  rabbit  by 
inoculation  of  blood  from  a  severe  case  of  Oroya  fever  fur- 
nishes no  support  to  the  hypothesis  that  other  vertebrates  act 
as  carriers  of  this  disease.  Upon  our  arrival  at  Lima  we  found 
the  Peruvian  government  entomologist,  Mr.  Charles  H. 
Townsend,  engaged  in  the  study  of  the  transmission  of  verruga 
peruviana  by  insects,  a  subject  which  he  had  pursued  for  some 
time  prior  to  our  visit.  His  conclusions  and  results  relating 
to  this  subject  will  be  considered  in  that  portion  of  the  report 
devoted  to  verruga  peruviana  (see  page  153).  We  regret  that 
we  did  not  have  an  opportunity  to  study  the  question  of  the 
transmission  of  Oroya  fever  either  properly  or  completely. 
The  question  is  obviously  one  of  great  importance  and  one 
which  should  be  thoroughly  and  carefully  pursued. 

Pathology 

Previous  Investigations. —  We  have  not  been  able  to  find  in 
the  literature  any  accurate  and  complete  description  of  the 
pathological  anatomical  lesions  of  Oroya  fever.  Evidently 
but  very  few  careful  and  complete  necropsies  have  been  per- 


PATHOLOGY  43 

formed  in  those  regions  where  the  disease  occurs  endemically. 
^Moreover,  the  disease  often  has  been  confused,  as  we  have 
emphasized,  with  other  diseases  which  occur  in  these  localities. 
Both  of  these  facts  tend  to  explain  the  absence  in  the  literature 
of  complete  and  accurate  descriptions  of  the  lesions  which 
occur  in  Oroya  fever.  In  the  estimable  monograph  of  Pro- 
fessor Odriozola  ^  we  find  the  most  complete  account  of  the 
gross  lesions,  and  Scheube,-  Plehn,"^  and  Castellani  ^  also  very 
briefly  allude  to  the  pathological  anatomy  of  the  disease. 
Odriozola  writing  upon  this  subject  states: 

"  Les  lesions  que  Ton  y  rencontre  sont  peu  considerables,  eu  egarcl  a  la 
gravite  de  la  maladie  et  aux  phenomenes  bruyants  cjui  I'accompagnent. 
Vn  fait  anatomo-pathologique  general  domine  I'etat  des  divers  organes, 
c'est  Vanemie.  Depuis  la  peau  jusqu'aux  profondeurs  de  I'organisme, 
tons  les  tissus  sont  pales,  exsangues.  Neanmoins,  les  complications  locales, 
congestives  ou  inflammatoires,  ne  sont  pas  rares  et  alterent  I'uniformite 
de  cette  paleur,  en  quelque  sorte  caracteristique.  II  est  en  effet  frequent 
de  voir  des  foA'ers  congestifs  ou  inflammatoires  du  cote  des  poumons, 
surtout  aux  bases;  de  meme,  on  peut  observer  une  congestion  elairsemee 
sur  la  musqueuse  intestinale,  ou  une  veritable  entente. 

"  Le  sang  est  tres  liquide,  se  coagule  tres  lentement  et  a  un  aspect 
noiratre.     Nous  en  reparlerons. 

"  Le  pericarde  contient  la  plupart  du  temps  une  quantite  de  liquide 
sereux  plus  ou  moins  considerable.  Les  cavites  du  coeur  sont  vides;  et 
on  y  trouve  tres  souvent  des  caillots  fibrineux  adherents  aux  parois  et  se 
prolongeant  j usque  dans  les  grands  vaisseux. 

"  Le  foie  et  la  rate  sont  frequemment  engorges.  Cette  hypertrophie 
portant  sur  le  foie  atteint  parfois  un  volume  tel  que,  pensant  a  une  hepatite 
suppuree,  on  a  pu  faire  de  grossieres  erreurs  de  diagnostic.  La  rate  peut 
egalement  augmenter  a  tel  point  ciu'elle  descend  presque  jusqu'a  la  fosse 
iliaque:  elle  peut  donner  I'illusion  de  la  cachexie  paludeenne.  Dans  ce 
dernier  cas  il.  est  surprenant  de  voir  ce  viscere,  une  fois  I'eruption  apparue, 
se  reduire  rapidement.  II  ne  faudrait  pas  croire  que  I'engorgement  de  ces 
deux  organes  soit  constant.  Nous  avons  constate  plusieurs  exemples  ou  la 
rate  ^tait  d'un  volume  normal  quelquefois  meme  reduit.  Mais  il  est 
Egalement  vrai  que  I'engorgement  du  foie  est  plus  constant  que  celui  de  la 

'  Odriozola:  ion.  c-it.,  p.  98. 

*  Scheube:   Die  Krankheiten  tier  Warmen  Lander,  Jena,  190.3,  416. 

'  Plehn:   Mense,  Handhueh  der  Tropenkrankheiten,  Leipz.,  190.5,  1st  ed.,  ii,  438. 

*  Castellani:  Castellani  &  Chalmers,  Manual  of  Troj)i('al  Medicine,  Lond.,  1913, 
2ded.,  1201. 


44  OROYA  FEVER 

rate:  nous  Tavons  enregistre  dans  presque  toutes  nos  observations.  La 
rate  se  presente  ramollie,  plus  ou  moins  friable.  Le  foie  offre  bien  souvent 
a  la  coupe  le  caractere  ardoise,  propre  a  Timpaludisme  et  qui  revele  la 
destruction  des  globules  rouges. 

"  Les  ganglions  lymphatiques  sont  totalement  engorges  et  cette  hyper- 
trophie  depasse  celle  xju'on  observe  dans  une  anemie  vulgaire;  ceux  du 
mesentere  surtout  acquierent  parfois  un  volume  comparable  a  celui  que 
Ton  trouve  dans  la  tuberculose  ou  dans  la  leucocythemie  ganglionnaire. 
Nous  regardons  I'engorgement  des  ganglions  lymphatiques  comme  un 
excellent  signe  diagnostique,  tres  appreciable  aux  regions  accessibles, 
telles  que  la  nuque,  la  region  sus-claviculaire  et  carotidienne,  les  aisselles, 
les  aines,  etc." 

Tamayo  ^  has  reported  upon  twenty-six  cases  of  Carrion's 
disease,  eight  of  which  were  fatal.  Necropsies  were  performed 
upon  five.  However,  almost  nothing  regarding  the  patho- 
logical conditions  observed  occurs  in  the  report.  In  three  of 
the  cases  it  is  stated  nothing  special  was  found  at  the  necropsy. 
In  the  other  two  the  autopsy  findings  are  reported  as  negative 
with  the  exception  of  the  fact  that  the  spleen  and  liver  were 
enlarged;  in  one  a  few  verrugas  were  found  upon  the  spleen 
when  there  had  been  no  eruption  upon  the  skin  during  life. 
Nevertheless,  the  blood  counts  made  upon  these  patients 
during  life  all  showed  very  advanced  anaemia,  so  that  some 
pathological  changes  must  have  been  visible  at  the  necropsies. 
No  report  upon  the  condition  of  the  intestines  or  other  organs 
is  given. 

Through  the  kindness  of  Dr.  Hercelles,  Professor  of  Path- 
ology in  Lima,  we  had  the  opportunity  of  examining  the 
pathological  material  relating  to  Oroya  fever  and  verruga 
peruviana  in  the  Museum  and  Laboratory  at  the  Dos  de  Mayo 
Hospital.  In  Dr.  Hercelles'  opinion  the  most  striking  lesions 
of  Oroya  fever  (cases  of  verruga  peruviana  without  the  erup- 
tion as  he  designated  them)  are  the  infarctions  in  the  spleen 
which  occur  very  commonly.  Infarctions  in  the  liver  he  has 
also  observed. 

Present  investigations.  —  Our  ideas  regarding  the  pathology 
of  Oroya  fever  which  follow,  are  based  largely  upon  the  study 

1  Tamayo:  Cron.  med.,  Lima,  1905,  xxii,  Nos.  406,  407;  J.  Trop.  Med.,  Lond.,  1906, 
ix,  159. 


PATHOLOGY  45 

of  organs  and  tissues  collected  by  ourselves,  but  the  material 
observed  in  and  received  from  this  institute  and  from  other 
Peruvian  physicians  has  also  been  considered  in  our  discussion 
of  this  subject.  The  latter  material,  however,  will  be  referred 
to  more  particularly  in  that  portion  of  the  report  deahng  with 
the  patholog}'  of  verruga  peruviana. 

The  most  striking  feature  observed  at  necropsy  of  patients 
which  have  died  with  Oroya  fever  is  the  evidence  of  an 
advanced  anaemia.  The  skin  shows  marked  pallor  and  is 
usually  of  a  pale,  j^ellowish,  waxy  color.  There  is  no  eruption 
on  the  skin  in  uncomphcated  cases.  The  conjunctivae  and 
finger  tips  are  also  pale.  Diffuse  and  punctate  hemorrhages 
may  occur  in  the  conjunctiva  and  in  the  mucosa  of  the  nose  or 
mouth.  The  superficial  lymphatic  glands  are  usually  moder- 
ately enlarged,  of  firm  consistence,  and  on  section,  of  a  pale 
pink  color.  Frequently  they  are  oedematous.  There  is 
almost  always  considerable  emaciation,  and  the  subcutaneous 
fat  is  scanty.  Small  punctate  hemorrhages  are  often  observed 
in  this  tissue.  A  few  petechial  hemorrhages  may  occur  in  the 
epicardium,  viscera,  pleura,  and  fat  elsewhere  in  the  body. 
These  are  not  extensive,  however.  There  is  a  tendency  to 
moderate  oedema  in  many  of  the  tissues.  The  heart  usually 
contains  partially  fluid,  pale  red  blood.  The  myocardium 
is  often  flabby;  a  few  small  discrete  ecchymoses  may  be 
present  in  the  pericardium.  The  lungs  are  usually  pale. 
They  sometimes  show  some  congestion;  more  commonly, 
however,  oedema  at  the  base.  A  few  petechiae  are  often 
observed  beneath  the  pleurae,  and  a  small  amount  of  pleural 
effusion  is  common.  The  spleen  is  usually  enlarged  and  of 
firm  consistence.  The  follicles  are  not  swollen  and  are  some- 
times indistinct.  Infarctions  are  common.  The  liver  is 
frequently  somewhat  swollen.  Its  consistence  is  flabby,  and 
the  cut  section  often  shows  yellowish  mottling  and  sometimes 
areas  in  which  necrosis  and  fatty  degeneration  is  perceptible. 
The  mesenteric  lymphatic  glands  are  swollen  and  on  section 
either  pale  pink  or  somewhat  reddened  in  color.  The  folHcles  of 
the  small  intestine  may  be  swollen,  but  in  other  cases  they  may 


46  OROYA  FEVER 

be  normal  in  appearance.  Superficial  ulcerations  have  been 
found  in  the  large  intestine.  Perhaps  lesions  of  this  nature 
develop  only  late  in  the  course  of  the  disease.  The  bone 
marrow  is  usually  distinctly  softer  than  normal,  and  the  sur- 
face sometimes  shows  a  grayish-red  mottling.  Carvallo  ^ 
has  found  an  increased  production  of  normoblasts  and  neu- 
trophilic myelocytes,  an  observation  which  we  have  also  been 
able  to  confirm.  The  necropsy  of  Case  15,  a  typical  one 
of  severe  Oroya  fever,  the  description  of  which  was  dic- 
tated at  the  time  the  necropsy  was  made,  illustrates  more 
in  detail  many  of  the  important  features  in  relation  to  the 
gross  lesions  encountered  in  this  disease,  and  is  therefore  in- 
serted here. 

Autopsy.  Case  15.  —  The  body  is  that  of  a  white  male,  Lithuanian,  with  light, 
dull  brown  hair.  The  body  measures  1  ra.  67.6  cm.  and  is  well  developed,  but 
sparely  nourished.  The  subcutaneous  fat  is  very  scanty.  The  skin  of  the  entire 
body  shows  marked  pallor,  and  is  of  a  pale  yellowish,  waxy  color.  Lips  and  finger 
tips  grayish  white.  The  conjunctiva  is  very  pale,  showing  numerous  diffuse  and  a 
few  punctate  hemorrhages,  and  also  injection  of  the  vessels.  The  latter  condition 
prevails  only  on  the  lower  lids,  the  conjunctiva  of  the  upper  lids  being  also  pale. 
Pupils  distended  and  equal.  The  skin  is  in  general  entirely  free  from  papules, 
nodules  or  tumors.  There  is  one  grayish  white  papule  on  the  right  forearm  over 
the  pronator  radii  teres  muscle  at  its  upper  third,  which  is  almost  the  color  of  the 
normal  skin,  and  measures  about  2  mm.  in  diameter;  it  is  very  slightly  raised. 
(Preserved  for  histological  study.)  There  is  no  other  lesion  noted  in  the  skin  of  the 
entire  body. 

Rigor  mortis  is  marked  in  both  upper  and  lower  extremities  and  there  is  moderate 
livor  mortis  over  the  dependent  parts ;  consisting  of  pale  red  blotches  on  the  dorsal 
aspect  of  the  trunk.  The  body  is  still  warm.  The  autopsy  is  commenced  two 
hours  after  death. 

On  stripping  the  pectoral  muscles  there  is  practically  no  visible  subcutaneous 
fat.  The  muscles  are  moist,  and  are  only  slightly  paler  than  normal.  The  sub- 
cutaneous and  intermuscular  fat  over  the  left  inguinal  region  shows  small  punctate 
hemorrhages  from  mere  points  to  1  mm.  in  diameter.  The  inguinal  lymph-nodes 
are  somewhat  enlarged,  of  firm  consistence  and  of  a  pale  pink  color.  The  marrow 
removed  from  the  femur  two  inches  above  the  median  portion  appears  distinctly 
softer  than  normal,  and  is  for  the  most  part  of  a  dark  red  color;  the  cut  surface 
shows  a  slight  mottling,  with  gray  in  certain  areas. 

On  opening  the  abdominal  cavity  the  peritoneum  is  smooth  and  shows  a  slight 
yellowish  tinge  over  the  surface  of  the  intestines.  The  appendix  is  long  and  bent 
upon  itself  behind  the  ileocecal  valve.  The  most  notable  abnormal  feature  is  the 
condition  of  the  spleen,  to  be  described  later  on. 

The  pleural  cavities  contain  a  small  amount  of  fluid.  There  are  no  adhesions. 
The  pericardial  sac  contains  a  small  amount  of  pale,  straw-colored  fluid,  possibly 
slightly  increased  in  amount.     The  epicardium  presents  numerous  small  discrete 

1  Carvallo:  These  de  Lima,  19n. 


PATHOLOGY  47 

ecchjanoses,  varying  from  mere  points  to  1  mm.  in  diameter.  These  are  most 
numerous  in  the  region  of  tlie  apex,  and  over  the  posterior  surface  of  the  heart. 

The  heart  is  rather  large  and  contains  a  small  amount  of  soft  clot,  but  much  pale 
red  blood,  which  distends  the  chambers.  The  heart  valves  appear  practically 
normal,  although  the  tricuspid  presents  several  soft  yellowish  thickenings  at  the 
border.  Above  the  aortic  valve  are  small  whitish  elevated  areas  somewhat  irreg- 
ular, and  often  connected  with  one  another.  The  intima  of  the  coronaries  is 
smooth.  Beneath  the  endocardium  of  the  left  ventricle  there  are  opaque  areas  of 
dull  grayish  color,  which  are  both  discrete  and  confluent,  and  give  the  surface  a 
mottled  appearance.  In  the  right  auricle  there  are  a  few  discrete  opaque  spots 
from  1  to  3  mm.  in  diameter.  The  myocardium  is  somewhat  flabby,  and  of  a  pale 
j-eUoAnsh  brown  color.  The  wall  of  the  left  ventricle  is  approximatelj'  1.5  to  1.8 
cm.  in  thickness. 

Lungs.  —  The  left  lung  contains  air  throughout,  and  there  are  no  adhesions  or 
palpable  nodules.  The  anterior  portion,  including  nearly  the  whole  of  the  upper 
lobe,  is  of  a  pink  gray  color,  mottled  \^^th  a  small  amount  of  black  pigmentation, 
while  the  remainder  of  the  lung  presents  a  great  number  of  small  petechiae  of  light 
red  color,  from  mere  points  to  2  or  3  mm.  in  diameter.  These  are  most  numerous 
over  the  dorsal  or  dependent  surface.  The  contiguous  borders  of  the  lobes  show 
a  small  amount  of  gelatinous  material,  possibly  exudate.  In  the  diaphragmatic 
surface  are  scattered  numerous  minute  elevations  of  dull  grayish  color,  not  per- 
fectlj^  rounded,  but  somewhat  irregular,  with  a  smooth  surface.  On  section  the 
cut  surface  reveals  no  macroscopical  lesions.  The  right  lung  contains  air  through- 
out, and  presents  practically  the  same  features  as  the  left,  the  petechiae  being  dis- 
tributed chiefly  over  the  dorsal  surface.  The  apices  of  both  lungs  are  free  from 
nodules  or  thickenings.  The  peribronchial  Ij^mph-nodes  are  not  notably  enlarged 
or  reddened  on  section,  although  somewhat  juicy. 

Spleen.  —  Capsule  smooth  and  tense;  the  surface  in  general  shows  bluish  color, 
with  irregular  patches  of  grayish  pink  beneath  the  capsule.  The  organ  measures 
17.7  X  10.1  X  7.6  centimeters.  On  section  the  cut  surface  appears  dark  reddish 
purple,  with  areas  of  dull  brown.  The  surface  is  slightly  uneven  except  in  several 
areas  to  be  described.  The  folUcles  are  not  \asible.  The  consistence  is  firm,  and 
on  scraping  the  cut  surface  only  a  small  amount  of  pulp  is  obtained.  There  are  a 
number  of  infarctions,  ha\ang  a  dry,  j^ellowish  gray  appearance.  None  of  these 
are  distinctly  wedge-shaped,  although  the  diameter  is  always  considerably  greater 
at  the  surface  of  the  spleen  than  in  the  spleen  substance.  A  few  are  roughly  tri- 
angular in  shape,  but  one  is  oval.  They  measure  from  2-3  mm.  to  3-4  cm.  at  the 
peripher>^  of  the  spleen. 

The  Liver.  —  Measures  27.9  cm.  X  19  cm  X  6.3  cm.  Capsule  is  smooth  and 
glistening,  \\'ith  a  few  irregular  scar-like  indentations  on  the  anterior  surface  in  the 
region  of  the  gall  bladder.  The  entire  surface  shows  whitish  translucent  markings 
of  a  branching  character,  as  though  marking  the  course  of  vessels  containing  pale 
reddish  blood.  The  cut  surface  of  the  liver  substance  presents  distinct  markings 
which  are  translucent  at  the  periphery,  so  that  a  net  work  of  pale  glistening  trans- 
lucent tissue  is  apparent:  included  within  this  is  a  yellowish  zone,  with  a  dull  red 
center.  The  consistence  of  the  liver  is  rather  flabby,  so  that  it  feels  soft  to  the 
hand,  but  it  is  slightly  tough  and  cuts  with  some  difficulty. 

The  gall  bladder  contains  golden  olive-colored  bile.  The  -pancreas  is  very  pale 
throughout,  and  presents  no  lesions. 

There  is  a  chain  of  enlarged  lymph-nodes  running  the  length  of  the  pancreas,  and 
situated  at  the  superior  border.  These  are  rounded,  dull  pink  in  color,  with  lobular 
markings.      On  section  they  arc  found  to  be  rather  soft  and  oedematous.     The 


48  OROYA  FEVER 

largest  is  situated  at  the  tail  of  the  pancreas,  and  is  red  in  color,  this  appearance 
evidently  resulting  from  hemorrhage. 

The  kidneys  are  rather  large  for  the  size  of  the  individual.  The  capsules  strip 
readily,  leaving  a  smooth  surface.  Both  kidneys  are  of  a  yellowish  gray  color  with 
very  pale  pyramids  and  papillae.  The  cortex  is  of  normal  thickness;  glomeruli 
barely  distinguishable.  There  are  no  hemorrhages  or  other  lesions  present.  The 
adrenals  are  thin,  and  present  no  abnormal  feature. 

The  ureters  are  normal.  The  bladder  contains  about  150  c.c.  of  urine.  The 
mucous  membrane  is  very  pale,  almost  white.  In  the  anterior  wall  are  two  ill- 
defined  thickenings  which  on  section  show  no  definite  lesion.  The  prostate  is  small, 
and  on  section  appears  of  almost  homogeneous  consistence,  and  of  a  whitish  gray 
color.     The  testes  show  no  lesions. 

The  stomach  contains  about  250  c.c.  of  thin  reddish  brown  fluid.  The  mucous 
membrane  is  a  dull  grayish  color,  with  shght  reddening  in  several  places. 

The  small  intestine  shows  no  macroscopial  lesions.  The  solitary  follicles  are  not 
swollen.  The  mucosa  of  the  large  intestine  is  generally  gray  in  color.  There  are 
very  numerous  superficial  ulcers,  which  are  brownish  red  in  color,  and  measure  3  or 
4  mm.  in  diameter.  Some  have  reddened,  irregular  borders,  others  are  round,  and 
with  slightly  undermined  edges.  There  are  a  few  Trichocephalus  trichiurus  in  the 
cecum. 

The  mesocolic  lymph-nodes  are  slightly  swollen  and  pink  in  color. 

The  ulcerations  noted  above,  scattered  throughout  the  large  intestine,  are  evi- 
dently in  the  heaUng  stage. 

The  aorta  throughout  its  length,  as  far  down  as  the  renal  veins,  shows  whitish 
irregular  elevations  of  a  reticulated  outline. 

The  head.  —  The  scalp  is  relatively  thick,  and  the  skull  is  from  7  to  9  mm.  in 
thickness.  The  cranium  is  of  a  rather  broad,  rounded  form.  The  dura  is  nowhere 
adherent  to  the  calvarium,  and  presents  nothing  abnormal,  with  the  exception  of  a 
lenticular  mass  of  pinkish  gray  color,  elevated  above  the  external  surface,  measuring 
2.5  to  3  mm.  in  diameter. 

The  blood-vessels  in  the  surface  of  the  brain  are  fiUed  with  pale  red  blood.  The 
surface  of  the  brain  is  rather  moist,  and  the  pia  is  somewhat  oedematous.  The 
convolutions  are  not  flattened.  On  removing  the  brain  there  is  considerable  fluid 
around  the  base,  and  escaping  from  around  the  cord.  This  is  of  a  clear  pale  yellow 
color.  The  lateral  ventricles  contain  only  a  small  amount,  about  5  c.c,  of  yellow 
fluid.     The  choroid  plexus  is  pale  yellow  and  gelatinous. 

Anatomical  Diagnosis. — -  Oroya  Fever.  Anaemia.  Petechial  hemorrhages  in 
epicardium,  viscera,  pleura,  and  inguinal  fat.  Ulcerations  large  intestine.  Slight 
acute  pleuritis  with  effusion.  Acute  splenic  hypertrophy.  Infarctions  of  spleen, 
Hyperplasia  and  congestion  of  lymph-nodes.  Active  bone  marrow.  Cloudy 
swelling  of  kidney.     Myocarditis.     Endarteritis  chiefly  of  aorta.     Oedema  of  pia. 

Microscopical  Examination  of  the  mucus  from  the  intestinal  ulcers  did  not 
reveal  any  amoebae  or  other  animal  parasites.  A  few  ova  of  Trichocephalus 
trichiurus  were  found  in  the  intestinal  contents.  The  bacteriological  examination 
of  the  cadaver  is  considered  on  page  39,  and  the  microscopical  examination  of 
smears  from  the  lymphatic  glands  and  spleen  on  page  33. 


:^:-'f^-^H:^^ 


':«i^^. 


Fig.  1.  —  Spleen.     Area  of  necrosis  at  holder  of  malpighiaii  nodule. 


Fin.  2.  —  Liver.     Showing;  area  of  necrosis,  central  type. 
Pl.\te  XII.  —  Oroya  Fever. 


'mw^^W^ 


■■^•«^i^-^i.-V'o?:^ 


...  V~' 


■^,':.  ■ 


O 


v^!^^ 
'^'$ 


•/■• 


■'i#S 


•^-  •,'*t'i'  ','»'  -.' :  i- 


Fig.  1.  —  Liver.     Stained  with  Scharlach  Roth.     Showing  areas  of  central  necrosis. 


Fio.  2.  —  LivKrt.     Showing  area  of  nccro.siw.     I''atty  dogciicration  of  hver  cells 

and  phagocytosis. 


Plate  XIII. — Ohoya  Fkvkk. 


HISTOLOGICAL   STUDIES  49 

HiSTOPATHOLOGY 

We  have  also  been  unable  to  find  any  accurate  report  of  the 
histological  changes  occurring  in  Oroya  fever,  or  indeed  anj'- 
account  of  the  specific  lesions  we  have  observed  in  the  micro- 
scopical study  of  the  tissues  of  individuals  dying  from  this 
disease.  Nevertheless,  in  tissues  presented  to  us  in  Lima  from 
Oroya  fever  cases  (designated  as  verruga  cases  in  which  there 
was  no  eruption  upon  the  skin),  we  have  sometimes  found  the 
same  lesions  we  have  observed  in  the  tissues  collected  at 
necropsy  by  ourselves.  The  tendency,  by  many  Peruvian 
phj^sicians  at  least,  has  been  to  regard  the  case  coming  to 
necropsy  with  no  lesions  of  verruga  either  upon  the  skin  or 
internally,  as  one  of  mistaken  diagnosis  or  as  one  of  verruga 
peruviana  in  which  death  has  occurred  before  the  nodular 
lesions  have  appeared.  The  interest  in  Peru  has  appeared 
almost  always  to  center  upon  the  histology  of  the  verrugas. 

Present  investigations.  In  the  histological  study  of  the  tissues 
from  persons  dying  from  Oroya  fever  we  have  found  marked 
changes  in  the  liver,  spleen,  bone  marrow,  and  lymphatic  glands. 

The  liver  shows  areas  of  toxic  degeneration  which  have 
apparently  resulted  from  the  activity  of  the  parasite  Barton- 
ella bacillifor7nis,  and  this  fact  would  suggest  that  during 
the  course  of  the  disease  in  addition  to  the  destruction  of  the 
red  cells  by  the  parasites  developing  in  them  another  patho- 
logical process  results  from  the  presence  of  a  toxin  in  the 
circulating  blood.  The  action  of  such  a  toxin  seems  to  be 
revealed  in  the  liver  by  the  presence  of  extensive  areas  of  ne- 
crosis of  the  central  type,  beginning  about  the  hepatic  veins. 
(Plate  XII,  Fig.  2.)  In  these  areas  many  of  the  liver  cells  in 
sections  hardened  in  Zenker's  solution  show  numerous  vacuoles 
in  their  cytoplasm  which  suggest  spaces  formerly  occupied 
by  fat,  and  in  tissues  hardened  in  formalin,  frozen  when  cut, 
and  stained  with  scharlach  Roth,  the  characteristic  stain  for 
fat  is  obtained  in  these  areas.  Distinct  zones  of  these  cells 
containing  fat  droplets  surround  central  areas  of  more  marked 
necrosis  in  which  many  of  the  liver  cells  are  very  granular. 


50  OROYA  FEVER 

Other  liver  cells  about  these  areas  appear  homogeneous  and 
hyaline  and  stain  intensely  with  eosin.  (Plate  xiii.)  Much 
granular  material  is  present  in  the  sinusoids  and  numerous 
swollen  endothelial  cells  and  endothelial  phagocytes  frequently 
containing  erythrocytes,  erythroblasts,  and  polymorphonu- 
clear leukocytes  are  present  in  the  periphery  of  the  necrotic 
areas  between  the  liver  cells.  (Plate  xvi,  Fig.  1.)  While  there 
is  not  extensive  pigmentation  of  the  liver,  moderate  amounts 
of  pigment  in  granules  or  small  masses  may  be  seen  within  the 
endothelial  phagocytes  and  in  a  few  endothelial  cells  lining  the 
sinusoids.  (Plate  xiv,  Fig.  2.)  Occasionally  small  granules  of 
pigment  may  be  seen  in  the  liver  cells.  Most  of  this  pigment 
does  not  give  the  iron  reaction;  it  is  yellowish  or  brownish  in 
color,  and  not  black.  Crystals  of  hematoidin  are  also  present. 
The  spleen  shows  numerous  infarctions.  In  sections  the 
edges  of  the  infarcted  areas  are  much  congested  and  show 
leukocytes  migrating  into  the  necrotic  tissue.  Many  of  the 
veins  show  thrombosis  suggesting  endothelial  injury  from 
some  toxic  or  infectious  agent.  At  the  periphery  of  many  of 
the  splenic  nodules  just  outside  the  capsule,  areas  of  necrosis 
are  visible  in  which  there  is  marked  deposition  of  fibrin. 
(Plate  XII,  Fig.  1.)  There  is  a  large  amount  of  pig- 
ment in  the  spleen,  which  occurs  in  small  or  larger  masses 
and  also  in  fine  granules.  (Plate  xv.)  It  is  yellowish 
or  yellowish-brown  in  color,  and  is  present  in  both  the 
endothelial  leukocytes  and  also  free  in  masses  between  the 
splenic  cells.  The  pigment  is  not  black,  and  does  not 
resemble  black  malarial  pigment.  It  resembles  melanin  in 
that  it  does  not  give  the  iron  reaction.  Whether  in  Oroya 
fever  the  anaemia  partially  results  from  the  destruction  of  the 
red  blood-cells  through  the  circulation  of  hematin,  as  W.  H. 
Brown  ^  believes  to  be  the  case  in  malaria,  at  the  present  time 
is  not  clear.  Bartonella  bacilliformis  evidently  produces .  no 
pigmentation  in  its  development  in  the  red  cells  in  the  periph- 
eral circulation.  No  malarial  parasites  are  visible  in  the 
splenic  sections  in  uncomplicated  cases.     The  spleen  is  rich 

1  Brown:  J.  Exper.  Med.,  1913,  xviii,  96. 


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Showing  necrosis  and  the  character  of  pigment. 


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Fig.  2 

Showing  endothelial  phagocytes  in  sinusoids,  enclosing 

erythrocytes,  erythroblasts,  polymorphonuclear 

leukocytes,  and  pigment. 


Plate  XIV  —  Oroya  Fever  Liver 


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HISTOPATHOLOGY  51 

in  pulp  and  much  congested;  many  nucleated  red  blood- 
corpuscles  are  visible.  Large  swollen  endothelial  cells,  some- 
times free,  at  others  attached  to  the  walls  of  the  blood-vessels, 
and  occasionally  containing  rounded  and  rod-shaped  elements 
are  observed.  Endothelial  phagocytes  frequently  contain- 
ing red  blood-cells  are  also  present  in  abundance.  In  the 
bone  marroio  there  is  also  striking  evidence  of  phagocytosis 
by  endothelial  leukocytes  of  the  red  corpuscles  in  various 
stages  of  development,  and  of  polymorphonuclear  leukocytes 
as  well  as  increased  production  of  normoblasts.  It  seems 
probable  that  this  extensive  phagocytosis  which  is  evident  in 
the  liver,  spleen,  and  bone  marrow  is  also  an  indication  of  the 
action  of  a  toxic  substance  which  has  led  to  chemical  changes 
or  injury  in  the  engulfed  cells. 

In  the  lymyhatic  glands  the  most  striking  pathological  pic- 
ture which  at  once  attracts  the  attention  is  the  presence  of 
large,  swollen,  endothelial  cells,  sometimes  free  in  the  lumen 
of  the  blood-vessel,  at  other  times  still  attached  to  the  walls. 
(Plates  VIII  and  ix.)  In  some  instances  a  single  endothelial  cell 
is  so  swollen  that  it  will  almost  occlude  the  entire  lumen  of  the 
vessel.  (Plate  xvi.  Fig.  2.)  These  cells  are  in  various  stages 
of  degeneration.  Many  of  them  are  distended  with  rounded 
and  rod-shaped  elements  and  it  is  apparent  that  a  multipli- 
cative phase  in  the  development  of  the  parasite  Bartonella 
bacilliformis  takes  place  within  these  cells.  (Plates  vi  and 
X.)  The  details  regarding  the  cycle  of  this  parasite  and 
the  changes  it  undergoes  in  these  cells  already  have  been 
referred  to  on  page  32. 

The  large  intestine  shows  ulcerations  with  distinct  under- 
mining of  the  mucosa  at  the  edges  so  that  frequently  the 
lesion  has  the  appearance  of  a  small  abscess  cavity  lying  in 
the  submucosa.  There  is  a  more  or  less  distinct  line  of  demar- 
cation of  the  abscess  with  vascular  granulation  tissue  forming 
the  wall;  numerous  polymorphonuclear  leukocytes,  fibrin, 
necrotic  tissue  and  bacteria  are  present.  In  some  places 
coagulation  necrosis  is  marked.  (Plate  xi,  Fig.  2.)  No 
amoebae  or  other  animal  parasites  are  visible  nor  does  the 


52  OROYA  FEVER 

ulcer  show  anything  histologically  characteristic  of  any  other 
disease.  The  blood-vessels  in  the  vicinity  of  the  lesions  show 
endothelial  cells  distended  with  the  rounded  and  rod-shaped 
parasites  already  described.  These  are  also  found  elsewhere 
in  the  blood-vessels,  of  the  submucosa  in  small  numbers  and 
in  the  lymphoid  tissue  of  the  submucosa  they  are  present  in 
large  numbers  where  they  sometimes  appear  to  obstruct  the 
blood-vessels.  It  appears  probable  that  this  may  be  the 
primary  factor  in  the  production  of  the  ulcerations,  and  that 
they  are  brought  about  in  a  somewhat  similar  manner  to  the 
ulcerations  of  the  intestine  sometimes  produced  by  Leish- 
mania  donovani  in  kala-azar  infection.  The  sections  of  the 
other  organs  reveal  no  changes  of  importance. 

We  have  said  that  we  have  been  unable  to  find  any  accurate 
account  of  the  histopathology  of  uncomplicated  cases  of 
Oroya  fever  in  the  literature.  In  an  instance  in  which 
sections  of  the  spleen  were  sent  to  Bindo  De  Vecchi,^  some- 
what similar  lesions  were  evidently  encountered  by  him. 

In  the  case  from  which  the  sections  came  there  were  no  nodules ;  also  the  clinical 
history  and  the  autopsy  made  no  mention  of  such  condition.  The  hemorrhagic 
lesions  of  the  spleen  were  very  striking.  These  began  in  the  follicles  and  finally 
destroyed  them.  The  liver  was  so  markedly  changed  that  the  tissue  could  hardly 
be  recognized.  The  sections  showed  so-called  "  dissociation  of  the  lobules  "  to  such 
a  degree  that  the  characteristics  of  the  cells  were  no  longer  present,  but  they  were 
massed  together.  The  single  cells  showed  different  lesions  of  degeneration  or 
necrosis  of  the  cell.  Also  different  liver  cells  showed  in  their  protoplasm  either 
single  or  multiple  inclusions  of  round  bodies  that  stained  deeply  with  a  basic  stain, 
with  a  ring  about  them,  and  often  accompanied  by  precipitated  pigment.  In  the 
markedly  changed  tissue  there  were  also  found  small  recent  hemorrhages  and  a  rich 
infiltration  of  leukocytes.  The  endothehal  cells  were  swollen  or  desquamated,  and 
some  of  them  assumed  the  function  of  phagocytes.  De  Vecchi  beheved  that  the 
inclusions  of  protoplasm  in  the  fiver  cells  were  to  be  regarded  in  all  probability  as 
the  remains  of  nuclei  from  leukocytes,  and  that  they  were  to  be  explained  through 
the  phagocytic  function  taken  over  by  the  fiver  ceUs  as  occurs  in  the  severe  degen- 
erative lesions  of  the  liver,  and  particularly  in  acute  yellow  atrophy. 

The  tissues  upon  which  these  descriptions  of  De  Vecchi  are 
based,  however,  were  sent  to  him  from  Peru,  and  he  had  been 
informed  that  the  patient  from  which  they  came  had  died  of 
severe  Carrion's  fever."  It  also  was  stated  that  no  verrugas 
of  the  skin  or  internal  organs  were  observed  at  the  necropsy, 

1  De  Vecchi:  Beiheft  4,  Arch.  f.  Schiffs-  u.  Tropenhyg.,  Leipz.,  1909,  xiii-,  143. 


4  • 


Fig.  1.  —  Liver.     Showing  endothelial  phagocytes  in  sinusoid. 


Via.  2.  —  Lv.Mi'H-.NfjDE.     Showing  swollen  endothelial  cell  in  blood-vessel  wall. 
Plate  XVI.  —  Ohoya  rEVF:R. 


HISTOPATHOLOGY  53 

and  that  a  paratyphoid  bacilhis  had  been  isolated  from  the 
spleen.  Possibh'  the  organism  encountered  in  this  case  was  a 
species  of  paracolon  or  colon  bacillus,  and  a  terminal  invader 
in  a  case  of  Oroya  fever.  In  the  light  of  our  own  observations 
the  changes  described  in  the  sections  of  the  liver  by  De  Vecchi 
suggest  such  a  possibility. 

Histological  Examination  of  other  Tissues 

Just  prior  to  our  departure  from  Lima,  and  after  our  in- 
vestigations there  had  been  completed,  we  obtained  from  the 
Hygienic  Laboratory  and  from  the  Dos  de  Maj^o  Hospital 
blocks  of  tissue  from  necropsies  not  performed  by  ourselves. 
In  these  cases  a  diagnosis  of  Oroya  fever  or  of  verruga  peru- 
viana had  been  made  during  life.  We  were  unable  to  obtain 
complete  histories  of  these  cases.  In  some  it  was  stated  there 
had  been  verrugas  upon  the  skin  during  life,  or  in  the  viscera 
at  autopsy.  However,  our  experience  in  Peru  has  led  us  to 
believe  that  lesions  may  be  sometimes  referred  to  as  having 
been  due  to  verruga  peruviana  which  more  careful  investiga- 
tion showed  to  be  of  an  entirely  different  nature.  These 
tissues  have  been  sectioned  and  studied,  and  the  notes  made 
regarding  them  are  as  follows: 

Histological  Examination  of  Material  from  other  Cases  in 

WHICH  A  Diagnosis  of  Oroya  Fever  or  of  Verruga 

Peruviana  was  made  by  Peruvian  Physicians 

Case  A.     Clinical  Diagnosis,  Oroya  Fever. 

Liver.  —  Shows  uniformly  distributed  central  degeneration  with  vacuoles  in 
the  liver  cells  probably  representing  fat.  An  occasional  phagocyte  situated  in 
the  sinusoids  presents  a  few  golden-yellow  pigment  granules.  The  endotheUal 
cells  of  the  small  vessels  occasionally  show  the  parasites  which  have  been 
previou.sly  described  fully  in  this  report.  (See  page  33.)  These  are  not  prominent 
features  and  are  only  found  after  considerable  search.  They  are  more  numerous 
in  the  small  vessels  of  the  .sinusoids  near  the  periphery  of  the  section  where  the 
preservation  is  more  perfect. 

Spleen. — Follicles  atrophied.  Several  show  relatively  large  areas  of  fibrinoid 
material  at  periphery.  Spleen  is  without  pigment.  Infected  endothelial  cells  are 
present  but  found  only  on  careful  search. 

Lymph-Nodes.  —  Show  no  marked  pathological  change  apart  from  the  swelling 
of  the  endothelial  cells  of  the  blood-vessels  which  are  distended  with  small  rod- 
shaped  bodies.    These  changes  are  found  more  frequently  in  the  small  veins. 


54  OROYA  FEVER 

Bone  Marrow.  —  Small  fragments  of  bone  marrow  consist  of  about  one-half  fat. 
There  is  considerable  phagocj^tosis  but  no  notable  lesion.  No  characteristic  changes 
are  found  in  the  endothelial  cells. 

Remarks. — This  is  undoubtedly  a  true  case  of  Oroya  fever  from  the  characteristic 
degeneration  of  the  liver  and  from  the  presence  of  Bartonella  bacilliformis  in  the 
Uver,  spleen,  and  lymph-node.     Note  that  there  is  practically  no  pigment  present. 

Case  B.     Clinical  Diagnosis,  Oroya  Fever.     (Formalin  fixation.) 

Liver.  —  Shows  a  large  amount  of  golden-brown  and  dark  pigment  distributed 
in  phagocytes  situated  in  sinusoids.  There  are  numerous  focal  lesions  consistent 
in  character  with  mihary  tubercles,  and  acid-fast  baciUi  are  found. 

Spleen.  —  Lymphoid  tissue  present  in  considerable  amount.  Small  amount  of 
yellowish-brown  pigment.     Focal  lesions  showing  acid-fast  bacilli. 

Remarks.  —  There  is  no  certainty  that  this  is  a  case  of  Oroya  fever.  Diagnosis : 
—  General  miliary  tuberculosis;  presence  of  malarial  pigment  questionable. 

Case  E.     Clinical  Diagnosis,  Oroya  Fever. 

Liver.  —  Extensive  central  degeneration  with  trabeculae  replaced  by  spaces 
filled  with  blood;  at  the  periphery  in  such  areas  there  are  the  remains  of  necrotic 
liver  cells,  still  further  out  the  liver  cells  are  for  the  most  part  vacuolated.  Oc- 
casional small  granules  of  pigment  are  found  in  phagocytic  cells. 

Lymph-Node.  —  A  mass  of  inflammatory  tissue  with  interior  portion  necrotic. 
There  are  giant-cells  at  the  periphery  and  smaller  foci  characteristic  of  tuberculosis. 
A  section  specially  stained;  shows  no  acid-fast  bacilli. 

Bits  of  brain  tissue  present  no  unusual  feature. 

Two  large  lesions  consisting  of  very  vascular  inflammatory  tissue. 

Remarks.  —  This  case  is  more  or  less  indefinite  in  several  respects.  The  liver  is 
interesting  in  that  it  suggests  a  later  stage  of  Oroya  fever,  possibly  one  bordering 
on  repair,  since  the  degenerated  parenchyma  has  completely  disappeared  in  the 
central  portion  of  lobules.  The  gland  is  probably  tuberculous  and  the  vascular 
inflammatory  tissue  is  probably  from  a  verruga  lesion. 

Case  W.     Clinical  Diagnosis,  Oroya  Fever.     (Preservation  poor.) 

Liver.  —  Generahzed  central  necrosis  with  vacuolation  of  surrounding  liver 
cells.  Small  amount  of  pigment  —  large  numbers  of  coarse  baciUi  distributed 
generally  throughout  the  tissue. 

Spleen.  —  Shows  a  small  amount  of  pigment.  Finer  changes  not  possible  to 
distinguish. 

Remarks. — This  case  shows  central  degeneration  of  the  hver,  and  a  small  amount 
of  pigment  in  this  organ  and  the  spleen.  Possibly  the  tissues  are  from  a  case  of 
Oroj^a  fever,  but  there  is  no  positive  evidence  of  this  from  the  histological  appear- 
ances. 

Case  X.  Clinical  Diagnosis,  Verruga  and  Oroya  Fever.  Viscera  fixed  in 
formalin. 

Liver.  —  Shows  focal  lesions  consistent  ^vith  tuberculosis,  also  a  considerable 
amount  of  golden-brown  pigment  within  phagocytes. 

Spleen.  —  Also  shows  focal  lesions  containing  acid-fast  baciUi.  There  is  a  small 
amount  of  pigment. 

A  Lymph-Node.  —  Shows  tuberculosis  but  no  changes  in  the  endothelium  such 
as  those  found  in  Oroya  fever.  Contains  peculiar  rounded,  oval,  or  long  dumb- 
bell-shaped bodies  included  within  phagocytes.  These  vary  from  reddish  purple 
to  almost  black. 

Skin.  — Very  vascular  tissue  covered  by  epithelium  consistent  with  verruga. 


HISTOPATHOLOGY  55 

Remarks.  —  General  miliary  tuberculosis,  possibly  verruga  lesions  of  the  skin. 
Xo  positive  evidence  of  Oroya  fever.  Presence  of  malarial  pigment  questionable. 
Peculiar  oval  bodies  in  a  tuberculous  lymph-node. 

Case  Y.  Clinical  Diagnosis,  Verruga,  Oroya  Fever,  and  Malaria.  Pre- 
ser^'ed  in  sublimate,  July,  1912. 

Liver.  —  Considerable  phagocytosis,  very  little  pigment.  Bacilli  distributed 
throughout  organ.     Xo  marked  degeneration. 

Spleen.  —  Practically  no  lymphoid  tissue  in  evidence.     Xo  pigment. 

Remarks. — The  amount  ot  material  is  scanty;  there  is  no  evidence  in  the  sections 
confirmatorj'  of  the  diagnoses  furnished  for  the  case. 

Case  Z.     Clinical  Diagnosis,  Verruga  Peruviana. 

Liver.  —  Poorly  preserved ;  almost  impossible  to  distinguish  any  pathological 
process. 

Spleen.  —  ]\Iuch  phagocytosis  and  occasional  pigment  granules. 

The  bone  marrow  shows  no  pathological  change. 

The  Skin  Lesiotis.  —  Are  composed  of  vascular  tissue  covered  over  with  epi- 
dermis.    Some  show  multiple  hemorrhages. 

Remarks.  —  There  is  enough  to  identify  the  latter  lesions  as  those  of  verruga 
peruviana,  but  the  preservation  of  the  tissue  is  such  that  it  is  impossible  to  form 
anj'  conclusions  as  to  the  e.xistence  of  Oroj^a  fever  in  this  case. 

Material  obtained  at  the  Dos  de  Mayo  Hospital  from  cases  diagnosed 
verruga  peruviana 

Specimen  from  Heart.  —  Shows  multiple  cysts.  Sections  show  that  these  are 
of  a  parasitic  nature  and  are  of  the  nature  of  cysticercus  cj'sts  (probably  C. 
cellulosae).     Xo  scolices  apparent.     Three  sections  examined. 

Specimen  from  Pharynx.  —  Situated  immediately  beneath  and  partially  destroy- 
ing the  epithelium  are  numerous  rounded  masses  of  vascular  tissue.  There  are 
also  large  masses  extending  somewhat  deeper.  There  is  no  tendency  to  caseation 
and  no  giant-cells.     Lesions  show  no  acid-fast  bacilli  in  specially  stained  sections. 

Remarks.  —  These  lesions  resemble  those  of  verruga  peruviana. 

Spleen.  —  Several  areas  of  degeneration  at  the  periphery  of  Ij^mph  follicles. 
There  is  considerable  dark  brown  pigment  which  from  its  distribution  suggests 
artefact.  There  are,  however,  a  few  granules  within  the  phagocytic  cells.  Xo 
characteristic  changes  found  within  endothelial  cells,  although  preservation  is  poor. 

The  results  of  the  study  of  these  tissues  are  interesting. 
From  the  description  we  have  already  given  in  this  report  of 
the  pathology  of  Oroj^a  fever,  it  is  evident  that  the  histology 
of  this  disease  is  pathognomonic;  and  we  see  in  the  study 
of  the  tissues  described  above  of  at  least  one  of  the  cases,  that 
a  definite  diagnosis  of  Oroya  fever  could  be  made  from  the 
presence  of  these  characteristic  lesions,  in  which  the  swollen 
endothelial  cells  containing  the  parasites  were  present.  As  has 
been  stated,  the  diagnosis  of  some  of  these  cases  given  to  us 
was  ''  verruga  peruviana,  with  Oroya  or  Carrion's  fever." 


56  OROYA  FEVER 

It  is  evident  that  cases  of  verruga  peruviana  have  been 
mistaken  for  cases  of  tuberculosis.  This  question  will  not  be 
considered  here,  but  will  be  discussed  more  fully  elsewhere  in 
this  report  in  relation  to  the  disease  verruga  peruviana.  (See 
page  138.)  Here  it  is  merely  desirable  to  call  attention  to 
the  fact  that  the  tissues  of  Cases  B  and  X  evidently  came  from 
patients  suffering  with  tuberculosis.  It  seems  evident  that 
the  tubercles  present  in  the  liver  in  these  cases  were  considered 
as  verrugas  of  the  miliary  type.  That  other  such  mistakes 
have  been  made  we  are  confident  from  the  fact  that  miliary 
tubercles  in  the  spleen  and  liver  due  to  Bacillus  tuberculosis 
have  been  shown  us  by  physicians  as  internal  verrugas.  It 
therefore  is  clear  that  in  Peru  miliary  tuberculosis  has  some- 
times been  diagnosed  incorrectly  not  only  as  verruga  peru- 
viana but  also  as  Oroya  fever.  Obviously  however  miliary 
tuberculosis  may  occur  as  a  complication  of  either  disease. 

The  distension  of  the  endothelial  cells  of  the  blood-vessels 
of  lymph-nodes,  spleen,  and  liver  of  Case  A,  and  the  presence  of 
large  numbers  of  the  parasites  demonstrate  that  these  tissues 
came  from  a  case  of  Oroya  fever  and  confirm  the  clinical 
diagnosis  of  the  disease  in  this  instance. 


IV 

VERRUGA  PERUVIANA 

Geographical  Distribution 

The  histor}^  of  the  disease  already  has  been  considered  in 
Section   ii.     From  this   account   it   is  evident   that  verruga 
peruviana  has  been  recognized  from  remote,  historical  periods 
in  Peru,  and  gradually  its  geographical  distribution  has  be- 
come well  defined.     So  far  as  is  known  the  disease  is  confined 
to  South  America.     It  is  certainly  more  commonly  found  on 
the  western  slopes  of  the  Andes  in  Peru.     However  it  seems 
hkely  that  cases  also  occur  in  southern  Ecuador,  parts  of 
Bolivia,  and  the  northern  part  of  Chili.     This  is  the  view  held 
by  Firth. ^     Zarate  in  1545  refers  to  the  occurrence  of  the 
disease  upon  the  Ecuadorian  coast,  and  Hall  ^  reported  a  case 
in  which  the  infection  was  contracted  in  Zaruma,  Ecuador. 
Formerly  it  was  stated   its  distribution  was  limited   in   the 
regions  in  Peru  between  9°  and  16°  south  latitude.     Today 
it  is  usually  said  to  be  confined  between  6°  and   13°  south 
latitude.     It  seems  possible,,  however,  that  verruga  peruviana 
is  related  to,  if  not  identical  with,  the  affection  described  by 
Bassewitz^  as  '^angiofibroma  cutis  circumscriptum  contagio- 
sum  "  which  he  found  to  occur  in  southern  Brazil.     If  this  is 
the  case  the  distribution  of  verruga  peruviana  must  be  still 
further  extended  to  20°  south  latitude.      Rocha-Lima '^  fol- 
lowing the  suggestion  of  Bassewitz  states  that  upon  clinical 
grounds  verruga  peruviana  and  angiofibroma  contagiosum  are 
probably   different   diseases,   since  in  verruga  peruviana  the 
eruption  is  preceded  by  an  acute  febrile  stage,  and  the  mortal- 

^  Firth:  Allbutt  and  Rolleston,  System  of  Medicine,  1912,  ii,  Part  2,  704. 
2  Hall:  Lancet,  Lond.,  1883,  Part  2,  845. 

^  Bassewitz:  Arch.  f.  Schiflfs-  u.  Tropenhyg.,  Leipz.,  1906,  x,  201. 
*  Rocha-Lima:    Verhandl.  d.  deutsch.  pathol.  Gesellsch.,  1913,  April,  409,  Gustav 
Fisher,  Jena. 

57 


58  VERRUGA  PERUVIANA 

ity  is  much  higher.  Rocha-Lima  has  evidently  accepted  the 
idea  that  Oroya  fever  and  verruga  peruviana  are  different 
stages  of  the  same  disease.  Since  he  has  apparently  never  seen 
a  case  of  Oroya  fever  and  but  one  case  of  verruga  peruviana, 
and  since  we  havejound  that  the  mortality  in  uncomplicated 
cases  of  verruga  peruviana  is  very  low,  and  that  the  fever 
preceding  the  eruption  is  rarely  severe  or  dangerous,  the 
clinical  manifestations,  urged  by  Rocha-Lima  as  distinguishing 
features,  between  verruga  peruviana  and  angiofibroma  con- 
tagiosum  are  not  apparent.  However,  further  investigation  is 
necessary  before  one  can  decide  definitely  upon  the  identity  of 
these  conditions.  So  far  extensive  inoculation  experiments  in 
animals  with  the  angiofibroma  contagiosum  have  not  been 
performed. 

Today  the  most  important  endemic  areas  of  verruga  peruvi- 
ana appear  to  be  located  in  the  narrow  valleys  on  the  western 
slopes  of  the  Peruvian  Andes,  locally  known  as  quehradas,  situa- 
ted at  an  altitude  of  from  2,800  to  9,000  feet.  (Plates  xvii  and 
XVIII.)  In  these  regions  the  disease  often  becomes  epidemic. 
In  the  wider  valleys  in  the  vicinity  it  seems  to  be  less  prevalent. 
The  localities  particularly  affected  have  in  general  the  same 
natural  features.  There  are  deeply  cleft,  narrow  valleys  whose 
sides  are  formed  of  comparatively  bare  rock,  consisting  largely  of 
granite  and  diorite,  and  are  almost  barren  of  vegetable  growth. 
Owing  to  the  absence  of  rain  during  the  greater  part  of  the  year, 
the  extremely  rocky  and  barren  character  of  the  soil,  and  the 
great  intensity  of  the  sun's  rays  during  the  day,  that  vegetation 
which  occurs  upon  the  slopes  consists  of  extremely  xerophytic 
types.  Conspicuous  among  these  are  cacti  of  several  kinds  in- 
cluding species  of  Opuntia  and  Cereus,  particularly  the  peculiar 
white-hooded  Cereus  senilis.  (Plate  xviii.)  A  fleshy  Euphor- 
bia with  cylindrical,  jointed  and  branched  stem,  resembling  an 
Opuntia  is  common  among  the  cacti  with  which  it  might  be 
confused  except  for  its  thin,  cylindrical,  deciduous  leaves.  A 
specimen  brought  to  Boston  later  developed  the  characteristic 
leaves  of  Euphorbia.  A  few  grasses  occur  on  the  higher  slopes 
but  all  are  dry  and  parched  in  the  drier  season  of  the  year. 


Fig.  1.  —  Regions  in  the  Andes  Mountains  in  which  Oroya  Fever,  Verruga 
Peruviana,  and  Malaria  are  prevalent. 


Fig.  2.  —  View  down  the  Rio  Rimac  from  above  Matucana,  showing  the  town. 

Plate  XVII.  — Peru. 


Fig.  1.  —  Type  of  Vegetation  on  the  lower  slopes  of  the  Canon 
CLOSE  TO  the  River  along  the  Trail  from  Surco  to  Matucana. 


Fig.  2.  —  Type  of  Vegetation  covering  the  higher  slopes  of 
the  Canon  along  the  Trail  from  Surco  to  Matucana. 


I'Ki.-i.  —  Oroya   Fever  limuGE  is  the  A.ndeh  Mountain.s. 
I'late  XVIII.  — Peru. 


GEOGRAPHICAL   DISTRIBUTION  59 

Trees  are  almost  entirely  absent,  although  a  very  few  scattered 
specimens  may  be  seen  here  and  there  far  up  the  slopes.  Cold 
mountain  streams  run  through  these  valleys,  the  soil  of  which 
is  formed  particularly  of  clay  which  is  covered  with  luxuriant 
vegetation  in  strong  contrast  to  that  on  the  slopes.  Odrio- 
zola  has  given  a  most  interesting  account  of  the  flora  in  these 
regions.  The  vegetation,  however,  is  abundant  only  in  the 
vicinity  of  the  streams  or  rivers,  or  where  irrigation  ditches 
have  been  constructed,  since,  as  intimated,  practically  very 
little  rain  falls  in  these  regions  the  greater  part  of  the  year. 
Here  and  there,  water  is  drawn  from  the  rivers  by  means  of 
sluiceways  and  carried  some  distance  down  the  valley  where 
it  is  used  to  irrigate  the  fields  that  occupy  the  nearly  level 
valley  between  the  steeply  rising  mountains  on  each  side.  As 
the  rivers  descend  rapidly  over  numerous  cascades,  plunging 
at  times  among  enormous  boulders  and  again  flowing  over 
short  gravelly  or  rocky  stretches,  there  is  little  vegetation 
along  the  immediate  edge  of  the  banks.  The  irrigated 
fields,  however,  are  extremely  fertile  and  numerous  cattle 
find  good  grazing  here  in  patches  of  grass  and  alfalfa. 
Just  above  these  there  is  a  semi-arid  level  with  numerous 
plants;  agaves,  leguminous  trees  resembling  Mimosa,  Pro- 
sopis  and  Parkinsonia,  and  a  great  variety  of  flowering 
herbs.  Maize  and  potatoes  are  grown  to  some  extent  in  the 
irrigated  areas,  but  no  other  crops  except  forage  plants  are 
extensively  cultivated.  Toward  the  lower  limit  of  the  verruga 
zone,  a  few  fruits  are  cultivated  such  as  oranges,  peaches,  and 
even  apples  in  addition  to  the  usual  tropical  fruits  like  the 
cherimoya,  granadilla,  avocado  pear,  etc.,  but  these  are  char- 
acteristic of  the  lower  level  rather  than  of  the  verruga  zone. 
Along  the  course  of  the  river  and  also  up  the  mountain  slopes 
on  the  edges  of  the  smaller  streams  that  rush  down  the  moun- 
tain side,  there  is  a  large  amount  of  pampas  grass  {Gynerium 
argenteum)  which  species  appears  to  be  quite  characteristic 
of  the  verruga  zone.  Likewise,  the  Prosopis-like  trees  and 
Mimosas  are  absent  from  the  lower  levels  where  the  place  of  the 
pampas  grass  is  taken  by  its  larger  congener,  Gynerium  gigan- 


60  VERRUGA   PERUVIANA 

teum.  It  would  thus  appear  that  the  verruga  zones  have,  by 
reason  probably  of  their  altitude,  a  flora  quite  noticeably 
different  from  Chosica  and  the  other  towns  just  below  the 
region  where  the  disease  occurs. 

The  climatic  conditions  in  these  valleys  are  also  peculiar 
and  different  from  those  of  Peru  in  general.  In  contrast  to 
the  rainless  coast  of  Peru,  there  is  what  might  be  called  a  rainy 
season  here,  even  though  the  rainfall  is  so  scanty.  The  disease 
appears  to  be  most  prevalent  in  the  summer,  and  particularly 
in  the  months  of  March  and  April  when  the  mountain  streams 
or  rivers  are  in  flood,  that  is,  about  the  close  of  the  rainy  season. 
The  days  in  these  vallej^s  are  hot  and  sunny,  and  the  tempera- 
ture may  reach  as  high  as  95°  to  103°  F.,  but  the  sun  sets 
behind  the  high  mountains  early  in  the  afternoon,  and  the 
nights  are  cold  and  during  them  the  direction  of  the  wind  is 
usually  reversed.  A  diurnal  variation  of  from  25°  to  35°  F., 
is  not  infrequently  observed.  Odriozola,  in  his  monograph  on 
verruga  peruviana,  gives  the  geographical  distribution  in  Peru 
up  to  1906  as  follows: 

In  the  Department  of  Lima  tKere  are  six  provinces  of  wliich  four,  Yauyos, 
Huarochiri,  Canta  and  Chancay,  constitute  endemic  areas  of  the  disease.  These 
provinces  contain  six  separate  river  systems,  and  include  nineteen  infected  com- 
munities, and  seven  additional  kno\vn  infected  but  uninhabited  localities.  The 
altitude  in  these  regions  ranges  between  2,900  and  7,900  feet.  In  the  Ancachs 
Department  the  provinces  of  Cajatambo,  Huaraz,  Huaylas  and  Pallasca,  the  last 
two  extending  into  the  southern  edge  of  Libertad  Department,  containing  four 
separate  river  systems,  are  also  infected,  showing  eight  isolated  valleys,  with  six- 
teen infected  communities,  the  altitude  of  these  districts  being  from  1,300  to 
10,900  feet. 

Townsend  ^  states  that  the  Magdalena  quebrada,  in  the 
department  of  Cajamarca,  has  very  recently  been  found  to  be  a 
strong  focus  of  verruga.  A  number  of  other  infected  localities 
have  been  discovered  since  the  publication  of  Dr.  Odriozola's 
monograph  and  further  investigation  has  shown  the  disease  to 
be  present  in  the  departments  of  Lima,  Ancachs,  Libertad, 
Lambayeque,  Cajamarca,  probably  the  southeastern  corner  of 
Piura,  and  perhaps  the  southwestern  part  of  Amazonas. 

1  Townsend:  J.  of  Economic  Entomology,  Phila.,  1913,  vi,  224. 


ETIOLOGY  61 

It  is  not  deemed  advisable  or  necessary  to  enumerate  in  this 
report  the  names  of  all  the  towns  and  villages  in  Peru  where  the 
disease  is  know^n  to  occur.  During  our  visit  to  Peru  the  most 
seriously  infected  areas  were  those  situated  in  the  vicinity  of 
the  railway  extending  from  Lima  to  Oroya,  in  the  territory  be- 
tween Chosica  with  an  altitude  of  2,800  feet  and  Matucana 
with  an  altitude  of  7,789  feet.  The  towns  particularly  in- 
fected at  this  time  were  Santa  Eulalia,  San  Bartolome,  Surco, 
Cocochacra  and  Matucana,  and  the  cases  w^e  studied  were 
discovered  largely  in  these  towns  or  their  vicinity,  or  in  the 
hospitals  in  Lima.  The  cases  we  observed  in  Lima  were  said 
to  have  contracted  the  disease  in  these  districts.  In  the  map 
given  by  Professor  Odriozola  in  his  monograph,  Matucana  is 
not  included  in  the  endemic  areas.  Cuesta  Blanca  with  an 
altitude  of  6,177  feet,  and  Surco  with  an  altitude  of  6,660  feet, 
are  the  points  with  the  highest  altitudes  given  as  endemic 
centers.  This  fact  shows  that  the  location  of  these  centers 
varies  somewhat  from  year  to  year.  We  were  informed  that 
the  disease  appeared  first  in  Matucana  in  epidemic  form  in 
March  and  April,  1912.  As  mentioned,  we  also  found  this 
locahty  infected  in  1913. 

Etiology 

Castellani  and  Chalmers,  writing  in  1913  in  their  ''  Manual 
of  Tropical  Medicine,"  say  the  causation  of  verruga  peruviana 
is  quite  unknown,  and  this  is  the  opinion  generally  accepted 
and  which  is  expressed  in  our  other  modern  textbooks  upon 
tropical  medicine  and  in  the  literature  generally  relating  to  this 
disease.  By  the  natives  verruga  peruviana  for  a  long  time 
was  supposed  to  be  contracted  by  drinking  water,  and  this 
view  was  accepted  by  Tschudi  ^  who  claimed  that  if  one 
avoided  drinking  from  the  suspected  springs  "  aguas  de  verru- 
gas "  they  escaped  the  disease.  Dounon  -  however,  first 
showed  that  there  was  no  basis  for  this  theory,  and  he  and  his 
assistants  all  drank  from  the  so-called  infected  springs  and 

'  Tschudi:  loc.  cit.  ^  Dounon:  loo.  cit.  * 


62  VERRUGA  PERUVIANA 

streams  without  contracting  the  disease.  Raimondi  ^  thought 
that  the  poison  of  the  malady  might  originate  from  venomous 
batrachia  which  hve  in  the  water,  or  that  the  disease  might  be 
due  to  a  virus  as  rabies  or  syphihs.  In  1875  Pancorvo  ^  called 
attention  to  the  fact  that  the  persons  who  usually  contracted 
it  worked  in  the  fields  or  were  brought  in  some  way  into 
intimate  contact  with  the  soil,  and  he  suggested  that  it 
was  an  intoxication  due  to  sulphureted  hydrogen  liberated 
from  the  earth.  This  view  never  received  marked  support, 
but  Firth  ^  in  1912  suggested  that  the  true  solution  of  the 
pathology  and  etiology  of  this  affection  wiU  be  found  in  the 
association  of  the  disease  with  some  form  of  parasitic  worm 
whose  free  stage  is  passed  either  in  water  or  mud,  most  likely 
in  mud.  Tupper  *  suggested  that  the  disease  was  transmitted 
by  a  miasmatic  agent.  Chastang  ^  called  attention  to  the  fact 
that  many  observers  consider  the  thorns  of  the  Cactus  opuntia 
to  be  the  cause  of  the  disease,  because  the  pricks  from  this 
plant  produce  cutaneous  lesions.  He  suggests  that  parasites 
clinging  to  the  thorns  may  be  the  cause. 

An  account  of  the  microorganisms  which  previously  have 
been  described  as  the  etiological  factor  of  verruga  peruviana  has 
already  been  given  in  Section  ii.  Page  10,  and  they  need  not  be 
referred  to  in  detail  here.  As  may  be  seen  from  this  account, 
Izquierdo,  Florez,  Odriozola,  Barton,  Biffi,  Tamayo,  Gastia- 
buru,  NicoUe,  LetuUe,  Escomel,  Galli  Valerio,  Mayer,  Rocha- 
Lima,  and  Werner  have  all  described  various  microorganisms 
as  the  cause  of  the  disease.  In  1913  ®  we  were  able  to  show 
that  verruga  peruviana  is  due  to  a  specific  virus  which  may 
be  transmitted  definitely  to  man  and  to  several  species  of  the 
lower  animals  by  direct  inoculation.  The  details  relating  to 
these  facts  will  be  referred  to  more  at  length  in  the  pages 
which  are  to  follow. 

1  Raimondi:  El  Peru,  1874. 

2  Pancorvo:  Gac.  med.,  Lima,  1875,  167. 

3  Firth:  Allbutt  and  Rolleston,  System  of  Medicine,  1912,  ii,  Part  2,  705. 
*  Tupper:  Ueber  die  Verruga  Peruviana,  Inaug.-Diss.,  Berlin,  1877. 

•  5  Chastang:  Arch,  de  med.  nav.,  1897,  417. 

6  J.  Am.  M.  Ass.,  Chicago,  1913,  Ixi,  1713. 


CLINICAL   DESCRIPTION  63 

It  is  generallj'  stated  in  the  textbooks  that  the  disease  is  not 
contagious,  and  in  this  statement  we  agree  in  that  it  certainly 
does  not  appear  to  be  markedly  contagious,  since  in  cases 
treated  in  the  general  wards  of  hospitals  no  tendency  is  ob- 
served for  it  to  spread.  It  is  also  generally  stated  that  the 
disease  is  transmitted  bj^  direct  inoculation,  and  that  this  was 
proven  by  Carrion's  experiment.  Although  we  have  the 
highest  admiration  for  the  courage  of  Carrion,  we  do  not 
believe  that  the  experiment  which  he  performed,  in  which  he 
inoculated  himself  with  the  blood  from  a  verruga  nodule  and 
died  subsequently  with  fever,  demonstrated  that  the  disease 
verruga  peruviana  is  inoculable.  No  lesions  developed  upon 
the  skin  in  Carrion's  case  according  to  the  reports,  but  no  one 
can  tell  today  definitely  of  what  he  died,  since  there  is  not 
dependable  and  sufficient  data  upon  the  subject.  As  men- 
tioned above,  however,  it  has  since  been  shown  that  the  virus 
of  the  disease  may  be  directly  transmitted  from  man  to  man, 
with  the  production  of  definite  lesions  and  without  fatal  result. 

Age,  sex,  and  race  apparently  have  no  influence  upon  the 
occurrence  of  verruga  peruviana.  We  observed  a  number  of 
cases  in  children  of  varying  ages,  and  in  both  male  and  female 
adults.  We  saw  cases  in  young  nursing  children,  and  Campo- 
donico  ^  and  Monge  ^  state  it  occurs  in  newly  born  infants. 
Xearlj^  every  one  who  lives  in  the  infected  areas  acquires  the 
disease  and  then  becomes  immune.  One  attack  usually  pro- 
tects. In  those  cases  in  which  reinfection  occurs  the  disease 
appears  in  mild  form.  Accession  or  relapses,  however,  during 
the  course  of  the  malady  are  not  uncommon.  Strangers  on 
visiting  and  remaining  for  any  length  of  time  in  the  endemic 
areas  usually  become  attacked.  It  has  been  repeatedly  stated 
in  the  literature  that  it  is  only  necessary  for  a  non-immune 
person  to  spend  a  single  night  or  to  sleep  in  an  infected  district 
in  order  to  acquire  the  disease.  In  regard  to  many  statements 
of  this  nature,  it  should  be  borne  in  mind  that  they  have  often 
been  made  in  reference  to  Oroya  or  Carrion's  fever.     Firth  ^ 

'  Campodonico:  Cr6ii  mod.,  Lima,  1895,  Feb.  15.  '  Firth:  loc.  cit. 

2  Monge:  J.  Lond.  School  Trop.  Med.,  1912,  Vol.  i,  Part  2,  164. 


64  VERRUGA  PERUVIANA 

points  out  that  prolonged  residence  in  the  endemic  centers  is 
certainly  not  necessary  to  contract  the  disease,  although  a 
mere  passage  through  the  country  without  either  eating  or 
drinking  on  the  journe.y,  or  being  thrown  in  intimate  contact 
with  the  inhabitants  is  not  sufficient  to  produce  it. 

Clinical  Features 

Incubation  Period. —  Castellani  states  the  incubation  period 
of  the  disease  is  not  definitely  known,  that  it  has  been  variously 
estimated  to  be  from  eight  to  forty  days,  but  is  most  usually 
from  twenty  to  thirty  daj^s.  Odriozola,  with  the  widest 
clinical  experience,  gives  the  incubation  period  from  fourteen 
to  forty  days.  Firth's  views  coincide  with  this,  and  we  have 
no  evidence  to  offer  to  the  contrary.  We  should  be  inclined 
to  regard  the  average  period  as  from  about  fourteen  to  twenty- 
one  days.  In  one  human  case  experimentally  inoculated  the 
period  which  ensued  between  the  inoculation  and  the  appear- 
ance of  lesions  was  sixteen  days.  In  monkeys  according  to 
our  experiments  the  incubation  period  usually  varies  from 
eleven  to  twenty-two  days;  in  one  experiment  of  Jadassohn  ^ 
it  was  sixty  days. 

Symptoms. —  It  is  generally  stated  in  textbooks  of  tropical 
medicine  that  there  are  three  clinical  stages  of  verruga  peru- 
viana: (1)  of  invasion;  (2)  of  eruption;  and  (3)  of  recovery. 
The  stage  of  invasion  in  our  cases  was  usually  characterized 
by  pain  in  the  joints,  the  knees,  elbows,  ankles,  and  wrists 
being  more  commonly  affected,  and  by  moderate  fever.  The 
temperature  sometimes  may  reach  104°  F.,  but  more  often  does 
not  exceed  100°.4  F.  The  fever  in  our  experience  ushers  in  the 
eruption  and  is  usually  of  short  duration,  lasting  sometimes 
but  a  few  days.  Other  observers  say  the  fever  may  last  a 
much  longer  time.  Castellani  gives  from  twenty  days  to  eight 
months;  Firth  from  one  to  nine  months  or  a  year.  Odriozola 
gives  the  average  period  of  fever  from  three  to  four  months. 
Malaria  is  such  a  very  common  disease  in  the  endemic  areas  of 
verruga  peruviana,  and  so  many  of  the  inhabitants  of  these 

^  Jadassohn  and  Seiffert:  Ztschr.  f.  Hyg.  u.  Infectionskrankh,  Leipz.,  1910,  Ixvi,  247. 


y 


./ 


Fig.  1.  —  Early  Miliary  Eruption. 


Fig.  2.  — Miliary  Eruption.     More  Advanced  Stage. 


Plate  XIX.  —  Verruga  Peruviana 


Fig.  3.  —  Eruption.     Most  Active  Stage  of  Disease. 


Fig.  4.  —  Euui'Tion.     Beginning  to  JIeceue 


(From  lyiiinicrc  I'lates;, 


Fig.  1  —  Nodular,  Subcutaneous,  and  Mulaire  Lesions. 


Fig.  2.  —  Nodular  amd  Mulaire  Lesions. 


Plate  XX.   -VERRuri.v  Peruviana  CFroin  Lumiorc  Plates). 


ERUPTION  65 

districts   are    infected   with  malaria  that    it  is  perhaps  not 
strange  that  these  statements  regarding  the  fever  vary  so  much. 
Thus  Scheube  ^  says  malaria  is  an  almost  inseparable  con- 
comitant of  verruga  and  bears  a  certain  relation  to  the  disease. 
Plehn^  also  refers  to  the  frequent  association  with  malaria 
and  states  that  the  fever  in  verruga  is  doubtless  sometimes  due 
to  malaria.     Hercelles  and  Gastiaburu  found  many  verruga 
cases  infected  with  malarial  parasites.     Patients  with  typhoid 
fever  and  Oroya  fever  in  these  districts  would  also  be  very 
hkely  to  contract  verruga.     Many  Peruvian  physicians,  among 
them  Odriozola,  admit  that  in  the  cases  of  verruga  peruviana 
not  preceded  by  the  Oroya  fever  stage,  the  fever  preceding 
the  eruption  is  of  a  brief  period  and  may  not  be  severe.     Castel- 
lani  says  slight  cases  may  pass  through  the  whole  illness  without 
fever,  while  more  severe  ones  may  show  intermittent  or  re- 
mittent fever;  this  is  also  our  experience  in  relation  to  the  fever 
in  this  disease.     Following  the  initial  fever  the  eruption  appears 
upon  the  skin  and  the  temperature  may  become  normal,  or  a 
slight  evening  rise,  of  a  degree  or  two,  may  persist  for  a  varying 
length  of  time.     Sometimes  during  the  period  of  invasion, 
particularly  at  the  onset,  there  is  complaint  of  some  headache 
and  backache.     The  physical  examination  of  the  chest  usually 
reveals   nothing   abnormal.     The   spleen   and   liver   are   not 
enlarged  in  uncomplicated  cases.     The  eruption  upon  the  skin 
which  is  the  characteristic  feature  of  the  disease  occurs  in  a 
variety  of  forms  according  to  the  stage  of  the  infection. 

Eruption. —  The  cutaneous  lesions  have  been  termed  "  mili- 
ary," "  nodular,"  or  ''  mulaire,"  according  to  their  size  and 
mode  of  development.  The  eruption  consists  at  first  of  ery- 
thematous areas  in  which  groups  of  small,  pin-point  sized  pap- 
ules soon  form.  Small  hemorrhagic  vesicles  also  often  appear 
which  may  be  grayish  or  reddish,  or  dark  blue,  according  to 
their  degree  of  vascularity  and  the  character  of  the  blood 
within  them.  (Plate  xix.)  The  areas  over  which  this  so-called 
''  miliary  "  rash  appears  are  frequently  oedematous,  and  this 

'  Scheube:  The  Diseases  of  Warm  Countries,  Lond.,  1903,  2d  ed.,  p.  299. 
^  Plehn:   Mense,  Tropenkrankhoitcn,  1905,  ii,  435. 


66  VERRUGA   PERUVIANA 

is  most  commonly  observed  on  the  legs,  and  more  rarely  upon 
the  extensor  surface  of  the  arms.  Unless  the  disease  is  arrested 
the  papules  grow  until  they  usually  measure  from  1  to  about 
5  mm.  in  diameter.  They  are  generally  reddened,  raised,  and 
sharply  circumscribed  from  the  surrounding  tissue,  more  often 
discrete,  sometimes '  confluent,  and  during  the  most  active 
process  of  the  disease,  are  of  a  bright  or  dark  cherry  color.  The 
skin  over  them  is  tense,  translucent,  and  adherent.  (Plate  xix.) 
They  are  at  this  time  usually  firm  or  cushiony  to  the  touch. 
When  injured  they  bleed  freely.  Later  in  the  disease  they 
assume  a  gray  color  or  become  the  color  of  the  skin.  As  the 
disease  advances,  in  addition  to  the  cutaneous  papules  deeper 
subcutaneous  nodules  may  appear,  which  at  first  are  frequently 
free  from  the  skin  and  lie  deep  in  the  subcutaneous  tissue. 
Later  many  of  these  grow  towards  the  surface  and  the  skin  over 
them  becomes  involved  and  adherent.  (Plate  xx.)  These  nod- 
ules vary  from  several  millimeters  to  three  or  four  centimeters 
in  diameter.  The  nodular  type  is  especially  common  about  the 
flexures  of  the  knees  and  elbows  and  over  the  thighs  and  legs. 
After  these  nodules  have  become  adherent  to  the  skin,  the  skin 
over  them  sometimes  becomes  broken  and  they  ma}''  ulcerate, 
reaching  the  surface  as  red  fungating  masses.  These  lesions  on 
becoming  protuberant  are  known  as  verrugas  of  the  ^'mu- 
laire  "  type  and  often  measure  several  centimeters  in  diameter. 
(Plates  XX,  and  xxi.)  They  also  may  be  pedunculated.  While 
the  eruption  occurs  most  commonly  on  the  legs  and  arms,  par- 
ticularly the  extensor  surfaces,  and  over  the  shoulders,  it  not 
infrequently  appears  on  the  face,  forehead,  and  ears.  Some- 
times it  appears  on  the  trunk  (Plate  xxiii)  and  sometimes 
upon  the  palms  of  the  hands  and  soles  of  the  feet.  A  better 
idea  of  the  extent  and  distribution  of  the  lesions  can  be 
obtained  from  the  illustrations.  (Plates  xix  to  xxiii.)  The 
miliary  eruption  also  may  occur  in  the  mucous  membrane  of 
the  mouth  or  nose,  or  upon  the  glans  penis.  Sometimes  when 
situated  in  the  nose  epistaxis  may  result.  After  lasting  for 
several  weeks  and  sometimes  for  three  or  four  months  or  even 
longer,   during  which  time  the   eruption  and  nodules  may 


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BLOOD   CHANGES  67 

disappear  and  reappear,  that  is,  accessions  or  relapses  occur, 
the  eruption  fades,  dries  up,  and  the  nodules  become  absorbed 
without  leaving  a  scar.  The  ulcerating  mulaire  lesions  heal 
by  cicatrization.  In  some  cases  only  the  miliary  eruption 
appears  and  convalescence  supervenes  after  several  weeks. 

The  opinion  is  often  expressed  that  the  eruption  is  often 
found  in  the  internal  organs,  and  that  there  exists  no  mucous 
membrane  or  organ  in  which  it  may  not  appear,  and  various 
symptoms  have  been  ascribed  to  its  presence  in  the  internal 
organs.  We  have  been  unable  to  find  sufficiently  definite 
evidence  for  these  general  conclusions,  and  it  appears  to  be 
one  of  those  statements  that  has  succeeded  from  one  author 
to  another  until  it  has  become  generally  accepted.  One  does 
find  in  the  early  literature  statements  regarding  the  occurrence 
of  the  verrugas  in  all  parts  of  the  body,  but  many  of  these  state- 
ments should  be  accepted  with  caution,  since  an  accurate 
knowledge  of  pathology  had  not  at  that  time  become  very 
widely  disseminated,  and  many  incorrect  diagnoses  of  lesions 
were  obviously  made.  Certainly  the  pathological  material 
preserved  in  Lima  relating  to  this  disease  does  not  give  this 
idea  of  the  distribution  of  the  lesions.  The  subject  will  be 
more  fully  discussed  when  the  pathology  of  the  disease  is 
considered. 

Blood.  —  Since  many  if  not  the  majority  of  cases  of  verruga 
have  previously  suffered  from  malaria,  it  is  not  unusual  to 
find  anaemia  present,  but  no  such  advanced  anaemia  exists 
as  has  been  observed  in  cases  of  Oroya  fever,  and  the  parasite 
of  Oroya  fever  is  not  found  in  the  blood-corpuscles  in  uncom- 
plicated cases.  Monge  ^  states  one  finds  a  marked  anaemia, 
but  that  enormous  differences  occur  in  the  number  of  the  red 
blood-corpuscles.  According  to  him  a  characteristic  feature 
of  the  disease  is  a  diminution  of  the  signs  of  the  anaemia 
from  the  time  the  patient  enters  the  hospital.  In  our  experi- 
ence in  well  marked  and  severe  cases  of  the  disease  the  red 
blood-corpuscles  did  not  number  usually  less  than  three  million. 
At  the  onset  of  the  eruption  the  leukocytes  are  sometimes 

1  Monge:  J.  Lond.  School  Trop.  Med.,  1912,  i,  Part  3,  239. 


68  VERRUGA  PERUVIANA 

increased,  but  usually  during  the  course  of  the  disease  they  are 
normal  or  reduced  in  number.  Monge  calls  attention  to  the 
fact  that  there  is  a  definite  mononuclear  increase  during  one 
stage  of  verruga.  The  following  table  shows  the  number  of 
red  and  white  cells  and  the  hemoglobin  estimation  in  several 
severe  cases  of  the  disease  in  the  eruptive  stage. 

Case    1.        Red  blood-corpuscles 3,900,000 

White  blood-corpuscles 14,000 

Hemoglobin 70  %  (Sahli) 

Red  blood-corpuscles 3,000,000 

White  blood-corpuscles 8,400 

Hemoglobin 75  %  (Sahli) 

Case    2.        Red  blood-corpuscles 3,000,000 

White  blood-corpuscles 4,400 

Hemoglobin 50  %  (Sahli) 

Case    3.        Red  blood-corpuscles 3,800,000 

White  blood-corpuscles 22,600 

Hemoglobin 55  %  (Sahli) 

Case  16.        Red  blood-corpuscles 3,000,000 

White  blood-corpuscles 4,200 

Hemoglobin 65  %  (Sahh) 

Case  17.         Red  blood-corpuscles 2,300,000 

White  blood-corpuscles 4,300 

Hemoglobin 55  %  (Sahli) 

Case  18.        Red  blood-corpuscles 1,600,000 

White  blood-corpuscles 4,500 

Hemoglobin 45  %  (Sahli) 

Case  19.        Red  blood-corpuscles 3,300,000 

White  blood-corpuscles 6,000 

Hemoglobin 45  %  (Sahli) 

The  differential  counts  on  these  cases  were  strikingly  differ- 
ent from  those  upon  Oroya  fever  (see  page  20).  The  follow- 
ing illustrations  are  taken  from  two  advanced  cases  of  verruga; 
in  one  the  white  count  was  normal,  while  in  the  other  a  leukocy- 
tosis was  present.  In  both  the  percentage  of  mononuclear  cells 
is  increased,  whereas  the  myelocytes  of  Oroya  fever  are  absent. 

Case  1  Case  2 

Polymorphonuclear  neutrophils 47  %  45  % 

Large  mononuclears 15  9 

Transitionals 8  4 

Lymphocytes 27  39 

Eosinophils 3  (2  cells) 

Unclassified —  2 


Plate  XXIII.  —  Verruga  Peruviana.     Showing  general  distribution  of 
nodular  lesions. 


ILLUSTRATIVE   CASES  69 

Prognosis. —  It  is  usually  stated  that  the  mortality  of 
verruga  peruviana  varies  between  10  and  40  per  cent,  but  this 
evidently  includes  cases  complicated  with  other  diseases.  In 
our  opinion  verruga  peruviana  when  uncomplicated  with  other 
diseases  is  very  rarely  fatal. 

Cases  illustrating  Special  Features 

The  histories  of  the  following  cases  selected  from  the  pro- 
tocols of  the  cases  studied  by  us  will  serve  to  emphasize 
many  of  the  cUnical  characteristics  of  the  disease. 

Case  1.  —  Extensive  Eruption;  miliary,  nodular,  and  mulaire  lesions. 
June  18,  1913.  Seen  in  hospital  in  Lima,  aged  eighteen  years,  lives  at 
Chosica,  but  has  recently  worked  for  long  periods  in  Santa  Eulalia.  Had 
tertian  malaria  six  months  ago.  Present  illness  of  three  months'  duration. 
No  fever  noticed  at  onset.  No  verruga  among  family  or  friends,  but  he 
has  been  associated  with  several  cases  in  his  work.  Patient  is  sitting  on 
bed,  looks  anxious,  slightly  dyspnoeic. 

Head. —  Eyes  equal,  movements  good  in  all  directions,  sclerae  clear. 
Pupils  equal,  react  to  light  and  accommodation.  Mucous  membranes 
very  pale.  Ears  are  free  from  tophi  and  discharge.  Teeth  are  rather  well 
preserved.  Tongue  very  slightly  coated.  Tonsils  not  enlarged.  Neck 
is  somewhat  thin.  Posterior  cervical  glands  are  not  palpable.  Both 
lobes  of  thyroid  are  palpable,  but  isthmus  is  not  felt.  There  is  slight 
fulness  over  the  right  parotid,  but  there  is  no  tenderness  on  pressure. 
Patient  complains  of  sensitiveness  and  pain  on  pressure  just  under  the 
ramus  of  the  left  maxilla. 

Both  epitrochlear  glands  are  markedly  enlarged.  They  would  appar- 
ently measure  about  1.5  to  2  cm.,  and  are  somewhat  sensitive  on  pressure. 

Chest  is  well  formed  and  rather  deep.  Respirations  are  shallow,  — thirty 
to  the  minute.  Respiratory  movements  equal  on  both  sides.  Vocal 
fremitus  better  felt  in  right  subclavicular  region  than  on  the  left.  Per- 
cussion note  slightly  dull  over  right  subclavicular  region  but  otherwise 
equal  over  fronts  and  axillae.  Breath  sounds  somewhat  harsh  over  right 
supra-  and  infraclavicular  regions.  No  rales  heard.  Fronts  and  axillae 
otherwise  clear. 

Heart. —  Point  of  maximum  impulse  best  seen  and  felt  at  mamilla. 
No  thrills  nor  shocks.  Relative  cardiac  dulness  extends  1  cm.  to  right 
of  sternum,  and  2  cm.  to  left  of  midclavicular  line.  Sounds  very  distant 
but  clear  at  apex.     Not  heard  over  aortic  and  pulmonic  areas. 

Abriomen.— Soft,  hepatic  and  splenic  dulness  is  not  increased,  and 
neither  the  liver  nor  spleen  is  palpable. 


70  VERRUGA  PERUVIANA 

Extremities. —  Deep  reflexes  are  active.     Romberg  negative. 
Blood  examination: 

June  22,  Wassermann  reaction  negative  to  beef  heart  antigen. 

June  27,  Red  blood-corpuscles 3,900,000 

White  blood-corpuscles 14,400 

Hemoglobin 70  %  (Sahh) 

July   19,  Red  blood-corpuscles 3,000,000 

White  blood-corpuscles 8,400 

Hemoglobin 75  %  (Sahli) 

July  22,  Wassermann  negative  to  four  antigens. 

The  examination  of  the  blood  reveals  no  parasites.  The  urine  is  normal. 
There  is  an  extensive  eruption  upon  the  skin. 

Cutaneous  Lesions. —  The  cutaneous  lesions  are  extensive  and  comprise 
lesions  of  the  miliary,  nodular,  and  mulaire  types. 

Distribution.  —  Lesions  occur  over  the  extensor  surfaces  of  hands,  fore- 
arms, and  arms.  (Palmar  surface  of  hands  and  flexor  surface  of  forearms 
and  upper  arms  clear.)  Forehead,  particularly  above  right  eyebrow, 
cheeks,  and  over  malar  regions.  A  few  pin-point  to  pin-head  sized  vesicles 
over  lips,  ears,  shoulders,  and  over  both  scapulae.  Back  and  trunk  gener- 
ally entirely  free  from  lesions.  Dorsal  surfaces  of  both  feet,  over  ankles, 
anterior  surface  of  the  tibiae,  and  right  thigh.  Also  on  the  posterior 
surface  of  the  left  thigh. 

Description  of  Lesions. —  Right  Arm,  Forearm. —  Entire  flexor  surface 
of  the  right  arm,  as  well  as  that  of  the  palm  of  the  hand,  is  entirely  free 
from  lesions.  On  the  extensor  surface  of  the  hand,  at  the  base  of  the  first 
and  second  fingers,  over  the  articulation  between  the  metacarpal  bones, 
are  groups  of  small,  discrete,  and  confluent  papules,  some  whitish  and  others 
pinkish  red  in  color,  measuring  usually  about  1  mm.  in  diameter,  and 
slightly  raised  above  the  surface  of  the  skin.  They  are  hard  to  the  touch 
and  have  a  rough  feeling  when  the  finger  is  rubbed  over  them.  Over  the 
wrist  there  is  a  papule  measuring  1.5  mm.  in  diameter,  and  raised  about 
1  mm.  above  the  surrounding  skin.  It  has  a  dome-hke  appearance,  is 
translucent,  and  pink  in  color.  It  is  sharply  circumscribed  from  the 
surrounding  skin.  Scattered  over  the  extensor  surface  of  the  whole  right 
arm  are  very  numerous  papules,  varying  from  pin-point  to  about  2  mm. 
in  size.  The  smallest  papules  are  grayish  white  in  color,  and  just  visibly 
raised  above  the  surface  of  the  skin;  those  that  are  more  slightly  raised 
and  slightly  larger  are  quite  hard  to  the  touch.  The  lesions  which  are 
slightly  larger  are  from  pink  to  bluish  red  in  color.  Some  are  the  exact 
shade  of  cherries.  These  are  translucent  in  appearance,  are  dome-like  in 
character,  sharply  circumscribed  from  the  surrounding  skin  and  discrete. 
They  appear  very  much  as  a  cherry  in  regard  to  translucence;  that  is, 
they  suggest  a  softer  consistence  beneath  their  perfectly  smooth  capsular 
membrane.     This  last  description  refers  to  those  areas  which  are  about 


ILLUSTRATIVE   CASES  71 

2  mm.  in  diameter.  They  are  raised  for  about  1.5  mm.  above  the  surround- 
ing skin,  and  are  quite  hard  to  the  touch.  Upon  pressure  of  some  of  the 
pinkish  lesions  they  become  grayish  in  color,  and  evidently  a  great  deal  of 
the  fluid  can  be  pressed  out  of  them.  Upon  pressure  on  others,  however, 
the  cherry-like  appearance  is  not  lost.  Some  of  the  smaller  lesions  appear 
almost  vesicular.  A  few  have  become  hemorrhagic  and  appear  as  dark  red 
points  on  the  skin,  measuring  about  1  mm.  in  diameter,  and  very  slightly 
raised. 

Over  the  bursa  of  the  right  elbow  is  a  nodular  area  measuring  2  cm. 
in  diameter,  and  about  1  cm.  in  height.  It  is  firm  to  the  touch  and  appears 
to  be  situated  outside  of  the  bursa. 

Over  the  back  of  the  arm,  and  on  its  exterior  aspect,  are  very  numerous 
papules,  similar  to  those  described  upon  the  forearm.  A  number  of  them, 
however,  are  dark  purple  or  black  in  color.  The  largest,  however,  does  not 
measure  more  than  2  mm.  The  darker  areas  evidently  represent  a  more 
advanced  stage  of  the  lesions.  The  progression  seems  to  be  from  the  small 
gray  pin-point  sized  lesions  to  the  pink  and  cherry-colored  ones ;  then  from 
the  resulting  hemorrhages  the  lesions  become  dark  purple  or  black,  and 
finally  dry  up,  leaving  blackish  scabs.  Beneath  some  of  the  blackish  scabs, 
however,  some  nodular  tumors  apparently  sometimes  form.  Upon  the 
right  arm,  over  the  insertion  of  the  deltoid  into  the  humerus  is  a  small 
nodular  tumor  of  this  character  measuring  5  mm.  in  diameter,  and  about 
1  mm.  in  height.  It  evidently  represents  a  small  verruga  of  the  nodular 
character.  The  lesions  over  the  right  shoulder  are  similar  to  those  de- 
scribed on  the  arm  and  forearm. 

Left  Arm,  Forearm,  and  Hand. —  Flexor  surface  and  palm  of  hand 
entirely  free  from  lesions,  with  the  exception  of  one  lesion  situated  on  the 
^vrist  at  the  base  of  the  thumb.  This  lesion  measures  13  mm.  in  diameter 
and  about  4  mm.  in  height.  It  is  moderately  firm  to  the  touch,  and  not 
freely  movable  beneath  the  skin.     Its  color  is  brownish  pink. 

Over  the  thumb  there  are  two  nodules  measuring  each  12  mm.  in 
diameter,  and  having  a  height  of  about  5  mm.  They  are  sharply  circum- 
scribed and  quite  firm  to  the  touch.  They  are  not  freely  movable  beneath 
the  skin.  They  are  almost  the  color  of  the  surrounding  skin,  but  slightly 
redder. 

Situated  over  the  third  finger,  at  the  distal  end  of  the  metacarpal  bone 
is  a  granulating  area  2  X  1.5  cm.  from  which  a  tumor  has  been  removed 
for  study. 

Over  the  wrist,  forearm  and  arm  are  numerous  miliary  lesions  similar 
to  those  described  on  the  right  arm,  but  they  are  not  so  numerous.  Over 
the  upper  third  of  the  arm,  on  its  posterior  surface  is  a  small  tumor  measur- 
ing 5  mm.  in  diameter  and  about  1  mm.  in  height,  which  is  grayish  blue  in 
color,  sharply  circumscribed  and  moderately  firm  to  the  touch.     When 


72  VERRUGA  PERUVIANA 

pressed  it  indents  slightly,  suggesting  that  its  contents  have  been  somewhat 
absorbed. 

Face. —  Over  the  forehead  there  are  scattered  very  numerous  grayish 
and  pinkish  mihary  lesions.  Over  the  right  eyebrow  are  a  group  of  dis- 
crete and  confluent  pinkish  and  cherry-colored  papules,  measuring  usually 
about  2  mm.  in  diameter.  Over  both  cheeks  there  are  numerous  mihary 
nodules,  one  on  the  right  cheek  having  reached  a  diameter  of  a  little  over 
2  mm.,  and  a  height  of  1  mm.  Over  the  right  inferior  maxilla  is  a  nodular 
tumor  measuring  4  mm.  in  diameter  and  2  mm.  high.  It  is  sharply  cir- 
cumscribed, moderately  firm  to  the  touch,  grayish  white  in  color,  in  the 
center  and  at  the  edges  grayish  brown.  On  the  conchae  of  both  ears  are 
numerous  lesions  of  the  miliary  type,  and  similar  to  those  already  described. 
They  are  much  more  marked  in  the  right  ear.  The  left  ear  contains  only 
fifteen  or  twenty  of  these  lesions.  Just  anteriorly  to  the  left  ear  is  a  small 
nodule  measuring  5  mm.  X  3  mm.,  and  about  1.5  mm.  in  height,  of  the 
nodular  type. 

Right  Leg. —  Over  the  anterior  surface  of  the  right  leg  and  foot  are  a 
number  of  lesions,  all  of  the  nodular  type.  No  miliary  lesions  are  any- 
where visible  on  the  leg  and  foot.  The  nodular  lesions  measure  from  6  mm. 
to  1  cm.  in  diameter.  Some  are  brownish  in  color,  others  brownish  red, 
and  some  have  a  pinkish  tinge.  There  is  one  measuring  1  cm.  in  diameter 
midway  over  the  third  metatarsal  bone.  Over  the  right  toe  is  a  granulat- 
ing area  1.5  cm.  in  diameter  from  which  a  tumor  has  been  removed.  There 
are  two  tumors  measuring  1  cm.  in  diameter,  one  over  the  external  and 
one  over  the  internal  malleoli,  and  there  are  four  more  small  tumors  over 
the  anterior  surface  of  the  tibia.  Two  of  these  seem  to  be  drying  up. 
Over  the  anterior  surface  of  the  tibia  7  cm.  above  the  ankle  joint  is  a  lesion 
of  the  so-called  mulaire  type.  Its  border  is  irregular  in  outline,  it  is  soft 
to  the  touch,  and  fluctuates  slightly.  Evidently  there  is  some  fluid  deep 
in  the  subcutaneous  tissue.  The  lesion  measures  about  2  cm.  in  diameter, 
but  it  is  not  sharply  circumscribed,  and  its  exact  diameter  cannot  be  stated. 

Over  the  right  thigh  above  the  knee  is  a  group  of  several  hundred  lesions 
of  the  miliary  type,  and  one  of  the  nodular  type,  measuring  1  cm.  in 
diameter,  and  2  mm.  in  height;  this  is  quite  firm  to  the  touch,  and  the  skin 
over  it  has  become  dry  and  whitish  gray  in  color.  The  tumor  itself  beneath 
the  skin  appears  of  a  dull  pink  color.  Posteriorly  over  the  soleus  muscle 
is  a  small  tumor  of  the  nodular  type  about  6  mm.  in  diameter.  It  is  the 
only  lesion  on  the  posterior  surface  of  the  leg  or  thigh.  The  sole  of  the  foot 
is  clear. 

Left  Leg. —  On  the  anterior  surface  of  the  thigh  there  are  numerous 
lesions  of  the  miliary  type.  All  of  the  nodular  type  are  below  the  left  knee. 
These  consist  of  seven  tumors  measuring  from  5  mm.  to  about  12  mm.  in 
diameter.     Several  have  been  removed  for  section.     These  are  all  sharply 


ILLUSTRATIVE   CASES  73 

circumscribed.  One  has  reached  a  height  of  nearly  half  a  centimeter  above 
the  surrounding  skin.  Over  most  of  them  the  skin  has  become  dried  and 
gra>dsh  white.  Just  below  the  external  malleolus  is  a  fungating  mushroom- 
like tumor  ^^^th  a  pedicle.  It  measures  2  cm.  in  diameter  at  the  top,  and 
is  raised  for  a  distance  of  1  cm.  above  the  surrounding  skin.  The  pedicle 
has  a  diameter  of  about  12  mm.  The  surface  is  in  places  grayish  and 
yellowish  in  color,  and  in  others  reddish.  This  variegated  appearance  has 
resulted  from  the  erosions.  This  lesion  is  of  the  mulaire  type.  On  the  leg 
there  is  posteriorly  one  nodular  area  over  the  middle  of  the  soleus  muscle; 
six  over  the  anterior  surface  of  the  leg,  and  one  over  the  dorsal  surface 
of  the  foot.  Over  the  middle  of  the  tibia  is  another  mulaire  lesion  measuring 
about  3  cm.  in  diameter,  and  not  sharply  circumscribed.  In  the  center 
there  is  slight  fluctuation.  The  edges  are  firmer  to  the  touch.  The  sole  of 
the  foot  is  clear. 

Laboratory  Examination.  —  Many  microscopical  preparations,  both  fresh 
and  stained,  were  made  from  the  lesions  of  the  skin  during  the  several 
weeks  that  the  case  was  under  observation.  Many  of  the  fresh  preparations 
were  examined  with  the  dark-field  microscope.  No  spirochaeta  or  other  rec- 
ognizable microorganism  was  found  present  either  by  microscopical  exami- 
nation or  by  animal  inoculation.  Attention  has  been  called  to  the  fact  that 
the  Wassermann  reaction  was  negative.  Numerous  cultures  and  animal 
inoculations  were  made  from  this  case.  The  notes  regarding  these  experi- 
ments will  be  found  on  pages  82  to  105. 

The  patient  under  generally  tonic  treatment  gradually  improved.  No 
fever  was  noted.  The  lesions  became  slowly  and  completely  absorbed, 
and  after  al)out  five  weeks'  stay  in  the  hospital  he  left  the  institution 
apparently  well. 

Case  17. —  Papular,  cutaneous,  and  subcutaneous  nodular  eruption. — 
July  10,  1913,  Male,  aged  eighteen  years.  This  patient  gives  a  very 
indefinite  and  unreliable  history.  He  lives  in  Lima  but  worked  in  Cococha- 
cra  two  days  in  February,  1912.  He  has  had  malaria  twice,  once  in  child- 
hood and  again  beginning  June  23,  1913.  He  had  a  short  attack  of  verruga 
fever  in  August,  1912,  lasting  two  weeks.  The  present  eruption  followed 
the  attack  of  malaria  and  rheumatism  in  June  of  the  present  year.  He 
does  not  know  of  any  association  with  verruga  patients. 

Examination. —  Conjunctivae  are  pale  in  color.  The  cervical  glands  are 
not  felt;  the  left  epitrochlear  gland  is  about  2  cm.  in  diameter.  Two  glands 
are  palpable  in  the  right  epitrochlear  region  each  about  1  cm.  in  diameter. 
The  inguinal  glands  are  very  large  and  firm.  From  the  history  given  they 
were  probably  involved  during  an  infection  with  Ducrey's  bacillus  about 
eighteen  months  ago.  The  examination  of  the  heart  and  lungs  reveals 
nothing  abnormal.     The  liver  is  not  enlarged.     The  spleen  is  firm  and 


74  VERRUGA  PERUVIANA 

extends  about  5  cm.  below  the  costal  margin.     The  urine  is  normal.     The 
blood  shows  no  parasites.     A  count  gives: 

Red  blood-corpuscles 2,300,000 

White  blood-corpuscles 4,300 

Hemoglobin 55  %  (Salili) 

The  Wassermann  reaction  is  negative. 

There  is  an  extensive  eruption  upon  the  skin. 

Distribution  of  Lesions. —  The  majority  of  the  visible  lesions  are  situated 
upon  the  lower  legs  and  feet.  There  are  a  small  number  above  the  knees, 
both  anteriorly  and  posteriorly,  and  there  are  two  small  nodules  on  the 
face  and  one  on  the  back  of  the  neck.  The  distribution  includes  the  skin 
over  the  dorsal  surface  of  the  feet,  the  posterior  surface  of  the  ankles, 
the  anterior  aspect  of  the  legs,  the  inner  aspect  of  the  calves,  the  knees, 
posteriorly  the  soleus  muscle  of  the  left  leg,  and  above  the  popliteal  space 
on  the  right  leg. 

The  lesions  consist  of  papules  and  cutaneous  nodules,  and  also  of  larger 
and  deeper  subcutaneous  nodules.  Some  of  the  subcutaneous  nodules 
also  involve  the  deeper  portion  of  the  skin.  The  papules  and  nodules 
situated  in  the  skin  vary  from  about  6  mm.  to  1.5  cm.  in  diameter.  They 
are  usually  raised  for  a  distance  of  0.5  to  1  cm.  above  the  surface  of  the 
surrounding  skin.  A  few  are  only  raised  about  2  mm.  and  these  have  a 
flattened  disc-like  appearance.  They  are  sharply  circumscribed,  and  a 
number  of  them  are  from  pink  to  cherry-colored,  while  others  are  brownish. 

Some  of  the  subcutaneous  nodules  are  larger  in  diameter,  several  measur- 
ing as  much  as  2.5  cm.  These  are  situated  particularly  above  the  knees  on 
both  legs,  and  on  the  exterior  surface  of  the  left  thigh.  They  usually  move 
with  the  skin,  and  their  margins  are  not  sharply  outlined. 

During  the  time  that  the  patient  remained  in  the  hospital  there  was 
little  fever.  The  evening  temperature  on  several  occasions  touched  100°.4 
F.  A  number  of  the  lesions  were  excised  from  time  to  time  for  histological 
study,  and  for  the  inoculation  of  animals,  and  numerous  cultures  were  taken. 
The  notes  regarding  these  experiments  will  be  found  on  page  110.  Many 
microscopical  preparations  both  fresh  and  stained  were  also  made  from  the 
lesions.  Some  of  the  fresh  preparations  were  examined  with  the  dark-field 
microscope.  No  spirochaetae,  acid-fast  bacilli,  or  other  visible  microor- 
ganisms were  detected  in  the  unbroken  lesions.  For  the  notes  of  these 
examinations,  see  pages  110  to  114. 

On  July  16,  the  note  regarding  the  case  was  made  as  follows:  "  There 
has  been  practically  no  fever.  The  lesions  seem  to  be  greatly  improved. 
Many  have  lost  their  cherry-like  color,  and  now  appear  either  pink  or 
grayish.  Some  are  beginning  to  be  brown.  They  also  look  less  tense 
than  formerly. 

"  The  patient  has  received  no  salvarsan  or  mercury  or  iodides;  nothing 
but  tonic  treatment." 


ILLUSTRATIVE   CASES  75 

On  July  29  the  note  reads:  "  Patient  is  in  greatly  improved  condition, 
and  indeed  one  might  say  almost  well.  Almost  all  of  the  nodules  and  all 
of  the  miliary  lesions  have  disappeared.  Situated  on  the  feet,  and  about 
the  ankles  posteriorly,  a  few  nodules  are  still  present.  On  the  dorsal  sur- 
face of  the  right  foot  is  a  tumor  which  is  pinkish  in  color,  and  which  meas- 
ures about  5  mm.  in  diameter  on  the  surface.  It  is  raised  for  about  2  mm. 
above  the  surface  of  the  skin.  This  was  removed.  On  section  it  was 
quite  moist  and  not  dried.  Another  nodule,  over  which  the  skin  has 
become  dried,  and  resembles  an  onion  skin,  measured  about  7  mm.  in 
diameter  and  5  mm.  in  height.  It  was  almost  the  color  of  the  normal  skin. 
This  nodule  was  also  removed.  On  cut  section  it  likewise  proved  to  be 
moist  in  the  interior." 

On  August  3  the  patient  left  the  hospital  apparently  well. 

Case  19.  —  Extensive  nodular  and  mulaire  lesions.  July  18,  1913. 
Male,  aged  twenty-nine  years.  Unmarried.  He  has  one  brother  who  is 
well;  parents  are  dead.  He  went  to  work  in  San  Bartolome  May  1  and 
worked  there  two  weeks.  After  working  at  San  Bartolome  he  returned  to 
Lima.  He  states  he  lived  recently  in  a  house  in  which  there  were  three 
cases  of  verruga.  There  is  an  eruption  upon  the  skin  which  he  says 
appeared  fifteen  days  ago,  first  on  the  right  wrist.  He  had  moderate  fever 
and  pains  in  the  joints  for  two  days  before  the  eruption  appeared. 

Examination.  —  The  mucous  membranes  are  pale.  The  sclerae  are 
clear.  The  left  posterior  cervical  glands  are  palpable.  The  examination 
of  the  heart  and  lungs  reveals  nothing  abnormal.  The  spleen  is  enlarged 
and  extends  8  mm.  below  the  costal  margin.  It  is  hard  and  firm.  The 
liver  is  not  enlarged.  The  blood  shows  no  parasites.  The  red  blood- 
corpuscles  number  3,300,000;  the  leukocytes,  6,000;  the  hemoglobin,  45% 
(Sahli).  The  Wassermann  reaction  is  negative.  The  temperature 
on  the  day  of  entrance  was  100°. 7  F.  There  is  an  extensive  eruption  upon 
the  skin. 

Distribution  and  Character  of  Lesions.  —  One  nodule  on  left  cheek  just 
anterior  to  the  ear,  and  one  on  lower  lip;  four  nodules  on  the  neck;  fourteen 
on  the  left  arm  and  forearm;  and  about  eighteen  on  the  right  arm  and 
forearm;  fourteen  over  the  shoulders  and  neck.  Three  nodules  on  the 
right  side  of  the  chest,  one  on  the  left  side  of  the  chest,  and  one  on  the 
anterior  surface  of  the  abdomen.  Seven  nodules  over  the  back.  In 
the  neighborhood  of  thirty  or  forty  nodules  on  the  legs  and  thighs. 

No  miliary  lesions  are  present.  The  nodules  generally  measure  from 
2  mm.  to  6  mm.  in  diameter.  A  few  are  larger.  These  measure  from  1  cm. 
to  1.5  cm.  in  diameter.  The  lesions  are  raised  from  2  mm.  above  the  sur- 
face of  the  skin  to  1  cm.  All  of  the  nodules,  except  a  few  on  the  feet,  are 
of  a  cherry  red  color.  They  are  sharply  circumscribed  and  all  are  discrete. 
In  addition  to  these  lesions  there  is  one  of  the  mulaire  type  measuring  12 


76  VERRUGA  PERUVIANA 

mm.  in  diameter  at  its  surface,  and  about  8  mm.  in  diameter  at  its  base. 
Clear  serum  is  exuding  from  this  lesion.  There  are  also  a  number  of 
subcutaneous  nodules.  One  of  these  is  situated  on  the  elbow  over  the 
head  of  the  radius,  and  is  not  attached  to  the  skin.  It  is  not  sharply- 
circumscribed  and  measures  about  0.5  cm.  in  diameter.  There  are  two 
similar  nodules  measuring  about  6  mm.  in  diameter  over  both  shoulders, 
and  two  more  slightly  larger  on  the  flexor  surface  of  the  left  forearm  and 
one  on  the  flexor  surface  of  the  right  forearm.  The  palms  of  the  hands  and 
soles  of  the  feet  are  free  from  lesions.  There  are  two  or  three  subcutaneous 
nodules  measuring  about  1  cm.  in  diameter  on  the  left  side  of  the  abdominal 
wall,  and  several  smaller  ones  to  the  right  side  of  the  rectus  muscle.  There 
are  numerous  subcutaneous  nodules  over  both  knees,  and  over  both  ankles. 
Some  of  the  nodules  in  these  localities  are  subcutaneous  and  some  cutaneous. 
Over  the  cutaneous  nodules  the  skin  may  appear  entirely  normal,  or  in 
those  instances  in  which  the  nodules  are  more  superficially  situated  the 
skin  has  a  slight  bluish  red  or  purplish  tinge.  The  inguinal  lymphatic 
glands  are  slightly  enlarged.  In  addition  to  these  lesions  there  are  three 
small  papules  (2  to  3  mm.)  on  the  mucous  membranes  of  the  lower  lip 
inside,  one  small  papule  2  mm.  in  diameter  on  the  gians  penis,  and  three 
or  four  of  similar  size  on  the  scrotum. 

Numerous  lesions  of  the  skin  were  removed  from  time  to  time  from  this 
patient  for  study,  and  fresh  and  stained  preparations  were  made  for  micro- 
scopical study.  No  spirochaetae,  acid-fast  bacilli,  or  other  microorganisms 
were  discovered  in  the  preparations  from  the  unbroken  lesions.  Some  of 
the  fresh  preparations  were  examined  b,y  the  dark-field  microscope.  For 
the  description  of  these  experiments  and  the  inoculation  of  animals  per- 
formed with  material  from  this  case,  see  page  114.  The  patient  gradually 
improved.  He  had  no  fever  during  his  stay  in  the  hospital.  He  dis- 
appeared from  the  hospital  towards  tne  end  of  July  before  the  lesions  had 
entirely  disappeared. 

In  some  cases  only  the  miliary  eruption  appears  upon  the 
skin,  and  the  disease  does  not  progress  to  the  nodular  stage. 
In  a  month  the  lesions  may  have  entirely  disappeared,  as 
occurred  in  the  following  case. 

Case  2.  —  June  20,  1913.  Male.  Aged  thirteen  years.  Worked  for 
six  months  in  Matucana  this  year.  None  of  his  family  are  living.  There 
is  one  case  of  verruga  in  the  house  in  which  he  lives.  The  present  illness 
began  in  May,  1913,  with  malaria  and  pains  in  the  joints,  followed  in  a  few 
weeks  with  the  eruption  of  verruga. 

Physical  Examination.  —  Mucous  membranes  somewhat  pale.  Cervi- 
cal glands  are  not  felt.  Epitrochlear  glands  enlarged,  measuring  about 
1  X  2  cm.     Tonsils    are  moderately  enlarged.     Heart  and  lungs  appear 


ILLUSTRATIVE  CASES  77 

normal.  Liver  is  not  enlarged.  Spleen  is  just  palpable  at  the  costal 
margin  and  is  hard  and  firm.  Inguinals  are  not  palpable.  The  urine  is 
normal.      The  blood  examination  shows  no  parasites;  the  blood  count 

shows 

Red  blood-corpuscles 3,000,000 

Leukocytes 4,400 

Hemoglobin 50  %  (Sahli) 

Wassermann  reaction  is  negative. 

There  is  an  extensive  miliary  eruption  upon  the  skin. 

General  Distn'hution  and  Description  of  the  Lesions.  —  Forehead,  chin, 
neck,  particularly  posteriorly,  shoulders,  arms,  forearms,  and  hands 
(trunk  entirely  clear),  thighs,  lower  legs  and  feet.  The  lesions  are  entirely 
miliary  in  type,  measuring  from  pin-point  to  2  mm.  in  diameter.  Over 
the  forehead,  neck  and  shoulders  they  are  grayish  white  in  color,  and  are 
evidently  receding. 

Both  surfaces  of  the  right  arm,  forearm,  and  hand  are  involved.  In 
the  palm  of  the  hand  the  majority  of  the  lesions  appear  as  small  hemor- 
rhagic spots  deep  in  the  skin.  Over  the  flexor  surface  of  the  forearm  the 
lesions  are  very  numerous,  and  almost  invariably  discrete.  They  consist 
of  grayish,  brownish  and  pinkish  papules.  On  the  extensor  surface  of  the 
hand  and  forearm  many  of  the  lesions  are  redder  in  color.  Some  are 
distinctly  blue.  On  the  back  of  the  hand  some  of  these  bluish  areas 
measure  as  much  as  6  mm.  in  diameter.  They  are  not  so  sharply  circum- 
scribed from  the  surrounding  skin  and  some  of  them  appear  to  be  situated 
beneath  it.  Over  the  flexor  surface  of  the  arm  and  extensor  surface  there 
are  numerous  grayish  white  papules. 

The  lesions  on  the  left  arm  and  forearm  are  similar  to  those  on  the  right. 
The  soles  of  both  feet  are  clear.  Over  the  dorsal  surface  of  both  feet,  an- 
terior and  posterior  surface  of  both  legs  and  thighs,  there  are  very  numerous 
papules.  These  are  grayish,  pinkish,  or  reddish  purple  in  color.  Although 
they  are  very  numerous,  and  frequently  closely  placed,  as  a  rule  they  are 
discrete.  On  pressure  they  do  not  lose  their  reddish  color.  Many  of  them 
appear  as  hemorrhages  beneath  the  skin.  The  majority,  however,  are 
distinctly  papular  in  character,  and  are  firm  to  the  touch,  and  give  a  rough 
feeling  when  the  hands  are  passed  over  them.  Over  the  internal  malleoli 
of  both  ankles  there  are  some  small  areas  measuring  about  0.5  cm.  in  diame- 
ter and  about  1  mm.  in  height,  which  are  grayish  pink  in  color.  These 
evidently  represent  confluent  papules.  There  are  also  some  other  areas 
over  both  tibiae  which  are  purplish  in  color,  and  which  have  very  irregular 
outlines.  These  evidently  represent  hemorrhages  which  have  occurred  in 
the  papules.  Many  of  these  areas  are  confluent.  On  pressure  most, 
though  not  all,  of  the  color  is  lost.  The  patient  remained  in  the  hospital 
several  weeks  during  which  time  he  had  only  an  occasional  rise  of  tempera- 


78  VERRUGA  PERUVIANA 

ture  of  a  degree  or  a  degree  and  a  half.  The  eruption  gradually  faded. 
No  nodules  appeared  upon  the  skin  or  beneath  it,  and  he  was  discharged 
apparently  well. 

In  children  the  disease  often  runs  a  mild  course,  and  the 
eruption  is  not  always  extensive.  The  child  may  not  appear 
ill  and  its  appetite  may  be  good  after  the  appearance  of  the 
eruption.  In  other  cases  a  more  copious  eruption  appears 
which  is  accompanied  by  fever.  Case  7,  described  on  page  107, 
and  the  following  cases,  Numbers  4  and  5,  illustrate  these 
features. 

Case  4.  — -A  child,  aged  fourteen  months,  seen  at  Santa  Ana  Hospital, 
Lima.  The  child  did  not  appear  ill  and  remained  contented  throughout 
the  examination.  The  physical  examination  shows  nothing  abnormal. 
There  are  a  number  of  lesions  on  the  skin.  The  glands  of  the  neck  are 
barely  palpable  in  the  region  of  the  tonsils  and  are  not  especially  enlarged. 
The  other  superficial  lymphatics  are  not  enlarged.  The  mucous  mem- 
branes appear  normal.  On  the  right  cheek  there  is  a  red,  slightly  raised 
papule,  measuring  2  mm.  in  diameter.  On  the  right  wrist  is  a  small 
reddish  brown  papule  4  mm.  in  diameter.  On  the  right  shoulder  is  another 
similar  papule.  On  the  right  leg  there  is  an  isolated  cherry  red,  hemi- 
spherical elevation  measuring  2  mm.  across,  and  1.5  mm.  in  height.  On 
the  inner  aspect  of  the  right  thigh  about  2  cm.  from  the  inguinal  furrow  is 
a  sharply  circumscribed,  raised  papule,  measuring  6  mm.  in  diameter, 
tense  to  the  touch,  and  somewhat  translucent.  This  lesion  was  later 
removed  with  the  scissors,  and  after  making  smear  preparations  the 
remainder  was  fixed  in  10  per  cent  formalin. 

On  the  anterior  surface  of  the  right  leg,  4  or  5  cm.  below  the  knee-joint, 
there  is  a  papule  measuring  2  mm.  in  diameter,  and  1  mm.  in  height, 
reddish  in  color,  with  the  overlying  skin  dry,  and  sHghtly  excoriated. 

On  the  outer  aspect  of  the  right  hip,  1  cm.  anterior  to  the  great  trochanter, 
there  is  a  papule  measuring  3  mm.  at  the  base,  tapering  cone-shaped  to 
1  mm.  at  top,  and  3  mm.  in  height.  This  is  pink  at  the  base,  and  of  a  dull 
red  to  brown  color  at  the  top.  It  is  hard  to  the  touch,  and  covered  by  a 
crust.  The  inguinal  glands  are  palpable,  of  the  size  of  small  peas.  In  the 
subcutaneous  tissue  on  the  outer  aspect  of  the  right  thigh,  2  cm.  above 
the  knee,  is  a  firm  nodule,  measuring  3  to  4  cm.  in  diameter.  It  is 
•freely  movable  beneath  the  skin. 

Examination  of  the  blood  is  negative  for  parasites. 

The  child  had  practically  no  fever  during  its  stay  of  about  one  month  in 
the  hospital.     It  took  its  food  well  and  was  discharged  without  lesions. 


ILLUSTRATIVE  CASES  79 

Case  5.  —  July  2,  1913.  Child,  aged  eighteen  months,  of  dusky  brown 
complexion.  This  child  is  evidently  ill,  and  whined  and  cried  throughout 
the  examination.  The  skin  is  hot  and  the  child  evidently  has  fever.  The 
mother  refuses  to  have  its  temperature  taken.  In  the  right  temporal 
region  is  a  rounded  tumor  fairly  well  circumscribed,  with  the  overlying 
skin  somewhat  reddened,  and  measuring  1.8  cm.  across.  Behind  the  right 
ear  is  a  rounded,  sharply  circumscribed  elevation,  6  mm.  across,  3  mm.  in 
height,  and  of  a  dusk>^  red  color.  On  the  left  arm  there  are  two  papules 
on  the  flexor  surface,  one  measuring  4  mm.,  the  other  2  mm.,  of  brownish 
red  color,  raised  1  to  1.5  mm.  above  the  surface.  On  the  forearm  there 
is  a  papule  measuring  3  mm.  in  diameter,  of  bright  red  color.  Just  above 
this  lesion  is  a  firm  nodule  beneath  the  skin,  measuring  5  mm.  in  diameter. 

The  right  forearm  shows  another  small  nodule  beneath  the  skin,  measur- 
ing about  4  mm.  in  diameter. 

Distributed  over  the  back  are  four  papules  of  bright  red  color,  slightly 
elevated,  and  measuring  from  1  to  3  mm.  in  diameter.  About  6  cm.  above 
the  umbilicus  is  a  small  red  papule,  measuring  3  mm.  across.  On  the  outer 
aspect  of  the  thigh  there  is  a  nodule  beneath  the  skin  about  1  cm.  in  diame- 
ter. The  right  foreleg  shows  about  nine  nodules;  not  all  are  discrete. 
These  are  not  sharply  circumscribed.  They  are  situated  beneath  the  skin, 
which  is  duskj^  red  over  the  central  portion  of  the  lesions,  and  the  color 
fades  gradually  and  irregularly  outward. 

The  sole  of  the  left  foot  shows  an  old  pinkish  red  papule,  measuring 
3  mm.  across,  with  the  overlying  skin  smooth  and  tense.  This  case  left 
the  hospital  before  further  observations  could  be  made.  We  learned 
subsequently  that  the  child  had  entirely  recovered. 

The  eruption  commonly  does  not  appear  upon  the  soles  of 
the  feet  or  palms  of  the  hands.  In  Case  2,  however,  it  was 
present  upon  the  palms  of  the  hands,  and  in  the  following  case 
upon  the  palms  and  particularly  upon  the  soles  of  the  feet. 

Case  9.  —  July  4,  1913.  Patient  fives  in  Callao,  and  comes  to  San 
Bartolome  every  Friday,  sleeps  there  and  returns  to  Lima  on  Saturda3^ 
He  has  done  this  since  last  November  with  the  exception  of  April  and  May, 
when  he  did  not  come  to  San  Bartolome  because  he  was  very  sick.  He 
commenced  to  feel  ill  on  the  30th  of  March.  At  that  time  he  had  high 
fever  and  pain  in  the  joints.  He  was  ill  in  bed  fifty  days  with  fever,  pains 
and  chills.  He  became  very  pale  and  suffered  with  vertigo.  On  the  16th 
of  May  the  eruption  appeared.  The  first  spot  appeared  on  the  left  wrist. 
Tills  lesion  now  appears  as  a  dried  wart  covered  by  a  blackish,  brown, 
dried  crust  measuring  3.5  mm.  in  diameter  and  2  mm.  in  height.  This  was 
removed,  microscopical  preparations  made,  and  placed  in  Zenker's  solution. 


80  VERRUGA  PERUVIANA 

The  remaining  eruption  consists  of  very  numerous  papules,  usually  discrete, 
scattered  over  the  extensor  surfaces  of  the  hands  and  forearms.  The 
palms  are  almost  free  from  papules.  There  are  four  in  the  right  palm. 
On  the  flexor  surface  of  the  wrists,  however,  the  papules  are  fairly  numerous, 
though  less  so  than  on  the  extensor  surface.  They  measure  from  pin-point 
to  3  mm.  in  diameter  and  are  pink  in  color.  They  are  very  slightly  raised, 
not  more  than  0.3  to  0.5  mm.  They  are  all  dry,  and  none  have  a  cherry- 
like appearance.  Over  many  of  them  the  epidermis  is  dry  and  desqua- 
mated, the  lesions  seem  to  be  receding. 

The  same  eruption  appears  on  the  soles  of  the  feet  as  well  as  on  the  dorsal 
surface  and  the  ankles.  None  above  the  middle  of  the  tibia.  On  the  feet 
the  lesions  are  more  hemorrhagic  in  character.  A  few  are  black  in  color 
from  the  resulting  hemorrhages.  Some  are  brownish  red  in  the  center, 
surrounded  by  pinkish  areas. 

As  we  have  seen,  great  variation  occurs  in  the  number  and 
size  of  the  excrescences  present  on  the  skin  in  the  different 
cases.  Sometimes  the  eruption  is  very  abundant,  as  in  Cases 
1,  17,  and  19,  and  sometimes  only  very  few  larger  lesions  are 
present.  In  the  following  case  but  a  single  lesion  was  present 
during  the  time  the  case  was  under  observation. 

Case  13.  —  Single  mulaire  lesion.  —  July  4,  1913.  A  woman,  aged 
twenty  years.  Lives  in  Matucana  where  she  was  seen.  Says  she  has  no 
children.  The  first  day  of  May  she  began  to  feel  sick.  Later  she  had  fever, 
became  anaemic,  and  had  pains  in  her  joints.  She  had  no  appetite.  The 
last  of  May  the  verruga  appeared.  Only  one  lesion  is  present  on  the  skin. 
This  consists  of  a  tumor  of  the  mulaire  type  situated  over  the  bursa  of  the 
left  elbow.  It  is  the  shape  of  a  chestnut,  and  measures  18  mm.  in  its  great- 
est diameter,  and  12  mm.  in  height.  It  is  of  a  dark  cherry  color,  and  firm  to 
the  touch.  The  patient  will  allow  no  specimens  to  be  made,  and  will  not 
allow  the  lesion  or  a  portion  of  it  to  be  removed.  Photographs  were  taken. 
Doctor  states  she  previously  had  some  nodules  beneath  the  skin  which  have 
now  disappeared.  She  has  no  fever  at  the  present  time.  There  is  very 
slight  anaemia. 

We  have  referred  in  other  parts  of  this  report  to  the  great 
prevalence  of  malaria  in  the  endemic  centers  of  verruga  peru- 
viana, and  to  the  fact  that  many  cases  of  verruga  peruviana 
have  either  previously  had  malaria  or  are  suffering  with  it  at 
the  time  the  eruption  of  verruga  appears.  So  common  is  a 
concomitant  infection  with  verruga  and  malaria  that  some 


EXPERIMENTS   RELATING   TO   THE  VIRUS  81 

authors  have  claimed  that  the  attack  of  verruga  predisposes  to 
the  malarial  attack  by  lowering  the  resistance  of  the  individual. 
In  the  following  case  w^hich  we  studied  there  was  infection  both 
with  verruga  peruviana  and  malaria,  and  the  febrile  infection 
had  been  regarded  as  the  Oroya  fever  stage  of  verruga. 

Case  14-  —  Verruga  Peruviana  complicated  with  Malaria.  —  July  14, 
1913.  Patient  lives  in  Matucana  where  he  was  seen.  Seven  yea,Ts  old. 
Born  in  Lima.  The  bo}^  arrived  here  last  October,  Disease  commenced  a 
month  ago.  Had  fever,  much  pain  in  the  joints  and  back  and  head.  Later 
he  became  anaemic.  His  temperature  on  the  first  day  was  102°. 2  F.  to 
103°.l  F.  in  the  afternoon;  101°.3  F.  in  the  morning;  today  it  is  103°. 3  F. 
At  present  there  is  oedema  of  the  legs  which  commenced  eight  days  ago. 
The  eruption  also  commenced  eight  days  ago.  The  first  lesion  was  situ- 
ated on  the  face.  At  present  there  are  numerous  miliary  lesions  scattered 
over  the  face,  legs,  forearms,  and  hands.  These  papules  measure  usually 
from  2  to  3  mm.  in  diameter,  are  raised  for  1  or  2  mm.  above  the  surface 
of  the  skin.  They  are  not  bright  cherry  in  color,  but  have  a  grayish  red 
appearance.  Also  mmaerous  subcutaneous  nodules  can  be  felt  beneath 
the  skin,  particularly  at  the  backs  of  the  wrists  and  hands,  and  above  the 
knees.  The  nodules  are  quite  firm  to  the  touch.  While  the  skin  is  raised 
over  these  deeper  nodules  it  appears  perfectly  normal.  His  sister  is  now 
in  Lima,  and  she,  his  mother  says,  also  has  verruga.  "  Her  doctor  thought 
she  had  malaria  from  the  fever,  but  last  Sunday  the  eruption  appeared." 
This  child  is  sick  in  bed  at  the  present  time. 

A  blood  smear  from  this  boy  shows  aestivo-autumnal  malarial  infection. 
Numerous  crescents  are  present. 

Experiments  Relating  to  the  Virus  of  Verruga 

Peruviana 

Transmission  to  animals  and  cultivation  of  the  Virus  of 
Verruga. —  Numerous  inoculation  experiments  were  made 
with  material  from  the  various  lesions  of  the  skin.  We  have 
already  referred  to  the  fact  that  we  were  unable  to  transmit 
the  parasite  of  Oroya  fever  to  animals  (see  page  34) ,  and  that 
intravenous  inoculations  of  the  blood  containing  the  parasites 
Bartonella  bacilliformis  into  monkeys  and  rabbits,  and  the 
intratesticular  inoculation  of  rabbits  produced  neither  local 
lesions,  verrugas  upon  the  skin,  nor  lesions  elsewhere.  The 
inoculations  performed  upon  animals  with  the  virus  of  verruga 


82  VERRUGA  PERUVIANA 

gave  positive  results  in  these  animals,  and  the  production  of 
definite  lesions,  which  in  the  case  of  monkeys  when  the  inocula- 
tion was  made  upon  the  skin  very  closely  resembled  those 
observed  in  the  human  cases  of  the  disease.  (Plates  xxiv 
and  XXV.) 

In  performing  these  inoculation  experiments  in  the  manner 
in  which  we  did,  several  objects  were  in  view:  first,  to  deter- 
mine if  the  disease  was  directly  inoculable;  second,  to  de- 
termine if  a  visible  microorganism  was  present  in  the  lesions; 
third,  to  determine  if  a  spirochaeta  was  present;  and  fourth, 
to  differentiate  the  disease  from  framboesia  and  syphilis.  In 
this  chapter,  therefore,  all  of  these  points  will  be  discussed 
more  or  less  widely. 

Frequently  a  portion  of  the  pathological  material  obtained 
from  the  human  verruga  cases  and  used  for  making  inocula- 
tions in  animals  was  also  employed  in  experiments  in  relation 
to  the  cultivation  of  the  virus  in  vitro,  and  sometimes  for  the 
determination  of  whether  the  virus  was  of  a  filterable  nature 
or  whether  its  presence  could  be  detected  by  the  microscope 
either  in  stained  smears  or  sections.  In  those  instances 
in  which  the  experiments  were  carried  out  with  the  same 
material  with  this  diverse  purpose,  it  is  more  convenient  to 
give  the  notes  regarding  them  just  as  they  were  made  at  the 
time  of  the  experiments.  The  results  obtained  in  relation  to 
each  of  these  problems,  however,  will  be  discussed  separately 
after  the  experiments  have  been  recounted. 

Experiments  with  Case  1 

June  20,  1913.  Patient  has  extensive  cutaneous  lesions  of  the  different 
types  of  the  disease,  including  those  of  the  miliary,  nodular  and  mulaire 
type.  After  cleansing  of  the  skin  with  soap  and  water,  alcohol,  ether  and 
iodin,  a  number  of  the  tumors  were  removed  for  examination  as  follows : 

Tumor  No.  1,  was  situated  on  the  inner  surface  of  the  left  ankle;  meas- 
ured about  1  cm.  in  diameter  and  6  mm.  in  height  above  the  surface  of  the 
skin,  and  was  of  the  nodular  type.  It  was  sharply  circumscribed  from  the 
surrounding  skin,  was  reddish  in  color,  and  of  rather  firm  consistence. 
The  skin  over  it  was  perfectly  smooth  and  rather  tense. 

Tumor  No.  2,  situated  on  the  extensor  surface  of  the  right  forearm; 
measured  about  3  mm.  in  diameter  and  was  raised  above  the  surface  of 


EXPERIMENTS  83 

the  skin  for  about  1  mm.  It  was  distinctly  red  in  color,  sharply  circum- 
scribed from  the  surrounding  skin,  and  of  the  miUary  type. 

Tumor  No.  3,  situated  on  the  extensor  surface  of  the  left  forearm,  of 
similar  character  and  appearance  to  Tumor  No.  2. 

Tumor  No.  4,  situated  over  the  dorsum  of  the  left  foot  just  below  the 
ankle  joint.  Measures  about  8  mm.  in  diameter,  and  projects  about 
4  mm.  above  the  surface  of  the  skin.  It  is  dark  red  in  color,  sharply 
circumscribed,  and  the  skin  is  smooth  and  rather  tense  over  it. 

Tumors  Nos.  1,  2,  and  3  were  removed  with  sterile  scissors,  and  tumor 
No.  4  with  a  sterile  knife,  some  of  the  normal  skin  being  excised  with  the 
last-mentioned  tumor.  Tumor  No.  2  was  placed  immediately  in  corrosive 
alcohol  solution  and  two-thirds  of  tumor  No.  4  into  Zenker's  solution. 

Cultures  and  Microscopical  Examination. — An  attempt  was  made  to 
aspirate  one  of  the  nodular  lesions  as  well  as  one  of  the  larger  lesions  of 
the  mulaire  type,  and  a  small  amount  of  blood  was  obtained  from  each  of 
these  lesions  which  was  inoculated  into  culture  media.  The  excised  tumors 
which  were  not  placed  in  hardening  solutions  were  put  in  sterile  Petri 
dishes,  and  brought  immediately  to  the  laboratory,  where  they  arrived 
about  fifteen  minutes  after  removal.  They  were  then  cut  into  numerous 
small  bits  with  sterile  forceps  and  scissors,  the  overlying  skin  being  not 
disturbed  and  many  of  these  bits  inoculated  into  test  tubes  containing 
pieces  of  rabbit's  testis,  kidney  and  in  a  few  instances  fat.  Ascitic  fluid 
was  then  added  to  all  of  the  tubes.  To  some  paraffin  oil  was  then  in 
addition  added,  and  to  others  alkaline  agar.  Some  of  the  tubes  were 
placed  in  sealed  jars  containing  phosphorus  under  strictly  anaerobic 
conditions.  In  all  twenty-eight  cultures  were  made,  twelve  of  which  were 
upon  agar. 

Many  microscopical  preparations  were  made  from  these  cultures  during 
the  next  three  weeks.  Some  were  examined  with  the  dark-field  microscope, 
others  were  stained  by  Giemsa's  or  Wright's  stains,  or  with  carbolfuchsin. 
No  spirochaetae  were  detected  or  other  microorganisms  which  we  regarded 
as  of  any  etiological  significance.  In  several  of  the  tubes  yeast  cells  from 
the  skin  developed,  and  in  several  others  either  cocci  or  coarse  bacilli. 
These  cultivations,  however,  were  only  exceptionally  found  in  the  tubes. 
With  these  exceptions  no  definite  or  constant  differences  were  observed  in 
the  appearance  between  the  inoculated  and  the  control  uninoculated  tubes. 
Smears  for  microscopical  examination  were  also  made  from  a  number  of 
the  pieces  of  the  tumors  which  remained;  some  of  these  were  immediately 
hardened  and  stained,  others  were  examined  with  the  dark-field  micro- 
scope. Rabbits  Nos.  1  to  7  and  Dog  No.  1  (q.  v.)  were  also  inoculated  with 
pieces  of  the  tumors.  Nothing  more  definite  than  motile  granules  was 
observed  in  the  preparations  examined  with  the  dark-field  microscope. 
In  preparations  stained  by  Giemsa  or  carbolfuchsin  no  spirochaetae  or 


84  VERRUGA  PERUVIANA 

other  microorganisms  were  discovered  except  a  few  yeast  cells  from  the 
skin  in  several  of  the  preparations. 

Inoculation  of  Animals 

Rabbit  No:  1.  — Testes  inoculation  June  20,  1913. 

Four  small  portions  of  the  tumor,  measuring  about  1.5  to  2  mm.  square,  were 
rubbed  up  in  2  c.c.  of  saline  solution  in  a  mortar,  and  0.3  c.c.  of  this  suspension 
inoculated  into  each  testis;    also  three  drops  subcutaneously  beneath  the  right 
side  of  the  scrotum.     Testes  measure  20  X  12  mm. 
June  21,     1913.  Testes  appear  normal. 

«     23.  Temp.  100°.0  F.     Testes  appear  normal  at  11  a.m. 

"     24.      4.30  P.M.     "       100°.2.  "        red,  no  longer  flabby. 

"     25.      4.43    "        "         99°.9..  "        red,  no  change. 

"     26.       5.10    "        "       100°.5.  "        red,  no  change. 

«     30.      2.00    "        "       100°.2.  "        unchanged  except  for  minute  dark 

spot  in  center  of  left,  and  in  right  testis 
small  just  palpable  nodule  on  anterior 
dorsal  surface. 
July     1.       5.05    "        "       101°.  Right  testis  definite  palpable  mass;  slight 

increase    over    yesterday.      Left   testis 
negative. 
"       3.      9.00  a.m.     "       101°.  Definite,  palpable,  firm  nodule  in  anterior 

portion  right  testis  not  over  a  few  mm. 
Left  testis  is  normal. 
«       5.      9.08    "       "       lOr.l.        Right  testis,  nodule  firm,  about  as  before. 

No  marked  increase.     Lesion  on  surface 
of  left.    Visible  but  not  palpable  at  site 
of  former  red  spot. 
7.      9.05    "       "        99°.8.        Right  testis,  definite  nodule  about  3-4 

mm.     Left  testis,  surface  shows  pale  red 
spot  2  mm.  diameter. 
"       9.     10.25    "        "       100°.  Right  testis,  firm  nodule,  no  marked  in- 

crease.   Left  testis,  spot  beneath  skin 
unchanged. 
"      11.     10.50    "       "        99°.4.        Right  testis,  nodule  firm,  3-4  mm.  ap- 
proximately.    Spot  on  surface  left  testis 
unchanged. 
"     13.      3.45    "  Right  testis,  firm  hard  nodule  3  mm.,  left, 

negative. 

Left,  negative  except  spot  on  surface. 
Right  testis,  hard.     Left,  negative  except 
minute  spot. 

Right  testis,  hard.  Left,  negative  except 
minute  spot. 
Smaller  and  harder. 
The  right  testis  shows  minute  translucent  mass  in  capsule. 
Left  testis  shows  a  firm  mass  in  its  interior.  There  is  a  bright  red  spot  in  pancreas. 
All  the  other  organs  appear  normal.  On  incision  of  the  testis  this  mass  is  found 
to  be  of  firm  consistence,  and  slightly  tough.  It  bulges  from  the  cut  surface  and 
has  a  slight  yellowish  tinge,  less  pink  and  transulcent  than  in  normal  tissue.  It 
is  somewhat  nodular,  and  has  a  sharply  circumcribed  outHne.  Measures  6X5  mm. 
Tissue  in  Zenker's  solution.     The  larger  lesion  photographed. 


«     15. 

10.00 

«     18. 

11.00 

"     21. 

10.40 

«     26. 

July  28. 

KiUe( 

EXPERIMENTS  85 

Cultures  on  agar  were  made  from  the  lesions;  these  were  still  negative  on  August 
1.  Microscopical  preparations  from  the  lesions  were  negative  for  spirochaetae  or 
other  microorganisms. 

Thirty-three  other  cultures  were  made  from  the  lesions,  small  bits  of  them  being 
placed  in  tubes  of  ascitic  fluid,  ascitic  fluid  +  rabbit's  testicle;  ascitic  agar  +  rab- 
bit's testicle;  ascitic  fluid  +  rabbit's  kidney;  ascitic  agar  +  rabbit's  kidney. 
One-half  of  the  liquid  cultures  were  covered  with  paraffin  oil.  An  equal  number  of 
control  tubes  were  prepared. 

Cultures  were  also  made  from  the  lesions  upon  blood-serum,  egg  media,  and 
blood  agar.  Many  microscopical  preparations  from  the  cultures  were  repeatedly 
examined  during  the  next  three  weeks,  some  with  the  dark-field  microscope,  others 
were  stained  ^\dth  Giemsa's  or  Wright's  stains,  or  with  carbolfuchsin.  No  spiro- 
chaetae or  other  microorganisms  were  found  except  in  one  tube  in  which  cocci  and 
another  in  which  bacilh  developed.  These  organisms,  however,  did  not  appear  in 
other  tubes  of  the  same  media  inoculated  in  the  same  way,  and  were  regarded  as  of 
no  special  significance.  In  the  examinations  made  with  the  dark -field  microscope 
very  numerous  motile  granules  were  of  course  usually  observed,  but  no  definite 
microorganisms  could  be  distinguished.  No  definite  and  constant  changes  between 
the  inoculated  tubes  and  many  of  the  control  ones  could  be  detected  during  three 
weelcs'  observation. 

Rabbit  No.  2.  —  Testes  inoculation.  June  20  inoculated  in  similar  manner  to 
Rabbit  No.  1,  except  no  injection  subcutaneously  in  the  scrotum  was  made.  Testes 
measure  21  X  12  mm. 

June  21,  1913.     Very  shght  subcutaneous  swelling  over  left  testis. 
"     23.  Temp.  100°.8  F.     Testes  appear  normal  at  11.05  .\.m. 

«     24.       4.24  p.m.      «       102°.8.  "  "  "      Still  soft. 

"     25.      4.50    "         «       102°.5.  "  «  «      Abscess  right  side 

of  jaw. 
"     26.       5.20    "         "       102°.6.        Testes  appear  normal.      Abscess  right 

side  of  jaw. 
"     30.      2.00    "        "       102°.6.        Left  testis,  definitely   palpable  nodule 

showing  increased   resistance   as   com- 
pared with  rest  of  testis.     Barely  pal- 
pable nodule  in  right  testis. 
July     1.       5.10    "         "       102°.7.        Both  testes  show  palpable  nodule  deep 

in  interior.     Increase  in  that  on  left. 
3.       9.07  A.M.      "       103°.l.         Large  rounded  nodule  on  jaw  about  2 

cm.  diameter.  Right  testis  shows  well 
defined  firm  nodule  several  mm.  in  diam- 
eter. Central  dorsal  portion  left  testis 
presents  a  larger  nodule,  outline  of  wliich 
is  becoming  well  defined. 
"       5.      9.20    "         «       102°.4.        Abscess  in  jaw  discharging.    Right  testis, 

nodule  well  defined,  larger.     Left  much 
larger,  surface  uneven. 
'•       9-15    "         "       100°.0.        Right  testis  shows  distinct  hard  nodule; 

remainder  of  testis  soft,  no  great  increase 

in  size.     Left  somewhat  harder. 

July  7.     Killed  by  ether  during  removal  of  testis.    The  left  showed  larger  nodule. 

This  measured  6  mm.  in  diameter,  bulged  from  cut  surface,  sharply  limited,  paler 

than  normal  surrounding  ti.ssue,  pale  pink,  translucent,  with  several  whiter  almost 

opaque  granules  in  cut  surface.     Right,  slightly  smaller  nodule  of  same  character, 


86  VERRUGA  PERUVIANA 

surface  of  both  slightly  uneven.  Smears  stained  with  Wright's  spirochaetae  stain  and 
carbolfuchsin  and  Gabbett  reveal  no  organisms.  Dark-field,  very  minute  poorly 
illuminated  dots  which  appeared  to  have  active  motion.  Portions  of  the  lesions  inoc- 
ulated by  trocar  into  two  rabbits'  testes,  and  others  ground  and  suspended  in  saline 
solution  and  injected  by  syringe  into  two  rabbits'  testes.  Rabbits  Nos.  28  to  32 
inclusive  inoculated  (q.  v.).  Cultures  from  the  lesions  were  sterile  after  3  weeks. 
Rabbit  No.  3.  —  Testes  inoculation.  Inoculated  in  the  same  way  as  Rabbit 
No.  2.  Inoculation  made  about  one  hour  after  the  removal  of  the  tumors. 
June  21,  1913.  Testes  appear  normal. 

"     23.  Temp.     99°.  1  F.     Testes  appear  normal  at  11.10  a.m. 

"     24.      4.30  P.M.      "       102°.l.  "        shghtly  redder  than  formerly. 

"     25.      4.45    "         "       lOr.3.  "        slightly  redder  than  at  first. 

«     26.       5.15    "         "       101°.9.  "        right  shghtly  tenser  than  left. 

"     30.       2.20    "         "       102°.2.  "        small,        uniform       consistency 

throughout  (fairly  firm),  except  minute 
indefinite  nodule  at  anterior  poles. 
July     1.       5.15    "         "       102°.4.        Neither  testis  shows  any  definite  thicken- 
ing. 
3.      9.12  a.m.      "       101°.3.        Barely  perceptible  nodule  middle  left 

testis.     Right  testis  normal. 
5.       9.30    "         "         99°.2.         Small,  barely  perceptible  nodule  left  tes- 
tis, about  middle. 
«       7.       9.22    "         "         99°.4.        Testes :  right,  small  hard  nodule  1  mm. 

.anteriorly.     Left,  larger  less  well  defined 
nodule. 
"       9.     10.25    «         "       101°.  Small  nodule,  right  testis,  shghtly  in- 

creased; firm  nodule  3  or  4  mm.  in  left 
testis. 
«     11.     11.00    "        "       ior.3.         Right  testis,  barely  distinguishable  un- 

evenness.     Left,  rather  ill-defined  nod- 
ule about  3-4  mm. 
"      13.      4.00  P.M.  Small  definite  hard  nodule,  anterior  dor- 

sal portion,  right,  and  firm  nodule,  left 
testis,  of  larger  size,  3-4  mm. 
July  14.  12.15  P.M.  Chloroformed.  Left  testis  photographed  after  section 
through  lesion  which  bulges  from  cut  surface.  It  is  firmer  than  the  surrounding 
tissue,  sharply  circumscribed,  and  pale  yellow;  homogeneous  and  slightly  opaque 
as  compared  with  the  translucent  pink  testis.  Right  testis  also  shows  a  lesion  on 
side  next  attachment  of  epididymis  of  similar  appearance  to  lesion  in  left  testis 
not  perfectly  rounded.  Right  in  corrosive  alcohol  2^  hours  later.  Left  in  10  per 
cent  formol  immediately.  Remainder  of  lesions  ground  in  mortar,  suspended  in 
2.5  c.c.  saline  solution,  and  injected  into  Rabbits  Nos.  44,  45,  46  (q.  v.).  Dark- 
field  examination  shows  motile  spermatozoa,  also  large  pairs  of  rounded  granules, 
numerous  indefinite  rods.  Microscopical  preparations  stained  with  Wright's  spiro- 
chaetal  stain  and  carbolfuchsin  negative  for  microorganisms.  Cultures  from  lesion 
in  each  testicle  on  agar  negative  after  fourteen  days. 

Rabbit  No.  4- —  Corneal  inoculation.     June  20,  right  cornea.     Two  furrows 
ploughed  in  surface.     Left  cornea  three  incisions.     Inoculated  with  tissue  from 
two  tumors  removed  from  Case  1  one  hour  previous  to  inoculation. 
June  21,  1913.     Surface  of  both  corneas  shghtly  uneven. 
"    23.  Temp.    99°. 6  F.    Surface  cornea  shows  slightest  possible 

unevenness  at  11.08  a.m. 


EXPERIMENTS  87 

June  24.      4.33  p.m.  Temp.  101°.9  F.     Surface  cornea  smooth, 
«    25.      4.50    "         "       100°.8. 
"    26.       5.30    "         "         99°.8. 

"    30.       2.25    "         "       ior.6.        Left    cornea    surface    smooth.      Right 

shows  three  or  four  shallow  pits. 
July     1.       3.23     "         "       101°.  Cornea  smooth. 

" '      3.      9.20  A.M.      "         99°. 7.  "        surface  smooth. 

«       5.       9.12    "         "         99°.  Two  definite  shallow  pits  in  the  right 

cornea. 
"       7.       9.25    "         "       100°.0.        Cornea,  right,  slight  unevenness  of  sur- 
face around  scratches. 
9.     10.30    "         "         99°.4.         Left  cornea  smooth.     Right,  smooth. 
"      11.     11.05    "  Both  corneas  smooth. 

"      15,     10.07    "  Both  corneas  smooth. 

July  18.     Corneas  smooth  nearly  healed.     Placed  with  stock  rabbits. 

Rabbit  No.  5.  —  Corneal  inoculation.  June  20  right  cornea,  two  furrows  ploughed 
on  surface.  Left  cornea,  three  incisions.  Inoculated  with  tissue  from  two  verrugas 
removed  from  Case  1  about  one  hour  previously. 

June  21,  1913.     Surface  of  both  corneas  slightly  uneven. 

Corneal  surface  smooth  at  11.12  a.m. 

Corneal  surface  smooth. 

«  »  (( 

Both  corneas  smooth. 


"     23. 

Temp. 

99°.S  F, 

«     24. 

4.36  p.m. 

« 

101°.8. 

"     25, 

4.56    " 

« 

102°.3. 

«     26. 

5.25    " 

(( 

103°.2. 

«     30. 

2.45    " 

u 

103°.l. 

July     1. 

5.25    " 

11 

102°.3. 

«       3. 

9.30  A.M. 

u 

101°.9. 

5. 

9.15    " 

u 

99°.5, 

7. 

9.30    " 

u 

102°.l. 

"       9. 

10.30    « 

« 

99°.7, 

"      11. 

11.05    « 

"      15. 

10.08    « 

July  18, 

Corneas  practically  healed. 

Placed  with  stock  rabbits. 

Rabbit  No.  6.  —  Intraperitoneal  inoculation. 

June  20,  1913.      Inoculated  fragments  of  verruga  nodule  from  Case  1  into 
peritoneal  cavity  by  perforating  near  umbilicus  with  a  trocar. 

June  23.     Temp.  99°.5  F.     Large  pustule  anterior  angle  left  eye  at  11.15  a.m. 
Temp.  101°. 8  F.     Abscess  on  eye  discharging. 


July 


24. 

4.48  P.M. 

25. 

5.03    " 

26. 

5.25    " 

30. 

2.50    " 

1. 

5.32    « 

3. 

9.35  a.m. 

5. 

9.35    " 

7.- 

9.35    " 

9. 

10.35    " 

11. 

11.05    « 

15. 

10.12  A.M 

100°. 

101°.5, 

102°.3. 

102°.  1. 

99°.8. 

Abdomen  soft. 

99°.7. 

(1                          u 

101°.3. 

u                   u 

100°.9. 

100°.4. 

100°.7. 

Killed.     Periton 

July  15.  10.12  a.m.  Temp.  100°. 7.  Killed.  Peritoneum  negative,  a  small 
amount  of  clear  fluid.  Smear  shows  a  few  degenerated  cells  and  phagocytes.  No 
tissue  saved.  Cultures  made  upon  agar  from  the  peritoneal  fluid  were  still  sterile 
on  July  27. 


88  VERRUGA  PERUVIANA 

RahUt  No.  7.  —  Cutaneous  and  subcutaneous  inoculation. 
June  20,  1913.     A  bit  of  verruga  nodule  implanted  by  means  of  trocar  near 
umbilicus,  and  another  near  right  nipple.    Two  scratches  in  skin  posterior  to  right 
nipple  inoculated  with  tissue  from  a  verruga  lesion  after  removal  of  hair. 

June  21.  Subcutaneous  implants  visible  beneath  skin.  Scarification  slightly- 
increased. 

June  23.  Temp.  100°.5  F.  Implants  near  umbilicus  and  near  nipple  barely 
palpable. 

June  24.     4.38  p.m.     Temp.  102°.0  F.     Subcutaneous  implants  if  anything 
increased  in  size.     Scarifications  diminishing. 
June  25.       5.05  p.m.  Temp.     98°.6  F. 
«     26.       5.30    "  "       102°.2.      Implants  still  palpable. 

«     30.      2.45    "  "       102°.8.  "  "  "  Implant  near 

nipple  shows  definite  increase  in  size. 
Cutaneous  scarifications  healed. 
July     1.       5.30    "  "       101°. 6.       Implants  about  as  yesterday. 

"       3.       9.30  A.M.        "         99°.2.       No  marked  increase;  larger  is  somewhat 

more  rounded.     Both  are  harder. 
"       5.       9.34    "  "         99°.6.       No  marked  increase  from  July  1. 

"       7.       9.35    "  "       101°. 5.       Subcutaneous   nodules   slightly   dimin- 

ished in  size,  rather  harder. 
"       9.     10.35    "  "       100°,9.       Implants  show  no  definite  increase. 

"      11.     11.00    "  Subcutaneous    implants    stationarj^    or 

diminishing  in  size. 
"      15.     10.10    "  "       lOr.9.       Slight  diminution  in  size. 

"      18.     11.15    "  Implants    distinctly    smaller.       Freely 

movable. 
"     21.     10.42    "  Implants    distinctly    smaller.       Freely 

movable. 
July  27,  1913.     Killed.     Larger  implant  7  X  4  X  2  mm.,  smaller  5  X  6  mm. 
yellow  gray,  larger  with  darker  gray  periphery.    Smear  of  smaller  stained  by  Giemsa 
and  carbolfuchsin  negative  for  microorganisms. 

Dog  No.  1.  Male.  —  Intraperitoneal  and  subcutaneous.  June  20,  1913,  inocu- 
lated subcutaneously  near  nipple,  left  side,  and  intraperitoneally  with  a  bit  of  verruga 
by  means  of  trocar.  Long  scratches  were  made  on  right  side  into  which  the  remain- 
ing verruga  tissue  was  thoroughly  rubbed. 

June  21,  1913.     Implant  palpable.     Scarification  healing. 
"     23.     11.20  A.M.  Temp.  101°  F.     Implant  barely  palpable.    Scarifications 

represented  by  linear  crusts.     Implant 
marked  by  spot.     Scarification  enclosed 
in  circle  of  carbolfuchsin. 
Implant   shows   no    change;     scarifica- 
tions practically  healed. 
Implant  to  left,  hard.     No  increase  in 
either.     Scarification  nearly  healed. 
Implant  diminishing  in  size. 
Implant,  no  increase. 


July 


24. 

4.45  P.M.  ': 

Femp 

.  101°.l. 

25. 

5.00    " 

« 

100°.5. 

26. 

« 

99°.5. 

30. 

2.55    « 

(( 

100°.9. 

1. 

2.35    " 

u 

99°.2. 

3. 

9.37  A.M. 

u 

99°.2. 

5. 

9.37    " 

" 

99°.9. 

7. 

9.37    « 

a 

100°.2. 

Possibl.y  slight     " 

Distinct  increase  in  implant. 

Implant  shows  distinct  increase  in  size. 


EXPERIMENTS  89 

July    9.     10.40  A.M.  Temp.     9S°.9F.    Subcutaneous  implant  is  increased   in 

size.     There  is  now  a  lens-shaped  mass 
measuring    about    8  mm.     across    and 
elevating  the  skin. 
"      11.     11.10    «  "       100°.2.       Implant  beneath  skin  9  mm.   appro.xi- 

mately. 
"      13.       4.00  P.M.  Mass  of  about  same  size,  firm. 

"      15.     10.15  A.M.        "       100°. 2.       Implant  shows  further  increase.     Less 

sharply  defined.  Overlying  skin  slightly 
thickened. 
July  16.  9.30  A.M.  Killed  with  chloroform.  A  flap  of  skin  dissected  back  to 
show  nodule  for  photographer.  It  lies  embedded  in  the  subcutaneous  fat  and 
appears  dark,  bright  red  with  pinkish  tissues  showing  through.  On  removal  it 
consists  of  a  flattened  lens-shaped  mass  measuring  1  X  1  X  0.25  cm.  in  diameter, 
is  firm  with  respect  to  retaining  shape;  on  pressure  thin  clear  fluid  exudes.  Grinds 
readily..  Axillary  lymph-node  on  left  side  is  larger  than  on  right  and  softer  and 
more  juicJ^  That  on  right  firmer  and  pigmented.  Right  8.5  X  8  X  1.8  mm. 
Left  10.3  X  10.2  X  4.5  mm.  Inguinal  not  found  on  careful  dissection.  Minute 
nodes  near  inguinal  ring.  Dog  had  been  castrated.  On  opening  the  peritoneal 
ca\'it}^  on  the  lateral  waU  a  few  cm.  from  median  line  the  peritoneal  surface  is 
somewhat  reddened  over  an  area  2.5  X  1.5  cm.  The  surface  of  this  area  is  studded 
with  small  translucent  red  nodules,  appearing  as  clear  drops  or  vesicles.  Some  of 
these  especially  at  periphery  of  area  are  scattered  and  discrete,  in  other  places 
closely  placed,  even  contiguous.  They  measure  from  mere  points  to  1  mm.  in 
diameter  and  strongly  suggest  in  appearance  the  miUary  lesions  often  seen  in  man. 
There  is  a  thickening  on  the  parietal  peritoneum  suggesting  point  of  puncture. 

The  spleen  near  the  posterior  ventral  end  presents  an  elevation  on  surface.  On 
section  a  rounded  mass  is  found  measuring  7X7  mm.  color  red  mottled  with  pale 
pink  with  a  row  of  small  nodules  or  follicles  (0.5  to  1.5  mm.)  at  periphery.  Edge  of 
subcutaneous  lesion  placed  in  Zenker's  solution.  Lymph-nodes  and  spleen  in  10 
per  cent  formol  and  Zenker's  solution.  Remainder  ground  after  2^  hours  in  salt 
solution  and  inoculated  subcutaneously  and  intraperitoneally  into  Dog  No.  3,  testes 
Dog  no.  2,  testes  Rabbit  no.  47.  Microscopical  preparations  and  cultures  from 
the  lesions  negative  for  microorganisms. 

Additional  Experiments,  Case  1.  —  On  June  25  the  patient  (Case  1) 
was  visited,  and  after  cleansing  the  skin  small  papules  measuring  2  to  3  mm. 
in  diameter  were  cut  or  scraped  off  with  scissors  from  the  face  and  arms. 
A  number  of  microscopical  smears  were  made  from  the  under  surface  of 
these  papules.  Fresh  preparations  were  also  made  from  their  cut  surfaces, 
and  one  of  the  papules  crushed  and  placed  beneath  a  cover  glass.  These 
fresh  preparations  were  examined  with  a  dark-field  microscope,  but  no 
definite  microorganism  could  be  detected.  Spirochaetae  were  particularly 
sought  for.  A  number  of  preparations  stained  by  Giemsa,  Wright,  or 
methylene  blue  were  studied.  In  some  of  these  preparations  blastomyces 
from  the  skin  were  fairly  common  and  occasionally  a  few  bacteria  were 
seen,  but  nothing  was  observed  which  suggested  a  specific  microorganism 
of  the  lesions.  Fourteen  cultures  were  made,  four  in  ascitic  fluid  contain- 
ing a  fragment  of  rabbit's  testis,  four  in  ascitic  fluid  agar  containing 


90  VERRUGA  PERUVIANA 

rabbit's  testis,  and  four  in  ascitic  fluid  agar  containing  rabbit's  kidney. 
These  media  were  inoculated  with  small  pieces  cut  from  the  interior  of  the 
papules.  Paraffin  oil  was  placed  on  the  surface  of  one-half  of  these  cul- 
tures. Corresponding  control  tubes  in  equal  number  were  prepared. 
Two  cultures  were  also  made  upon  ordinary  agar. 

On  July  21  the  note  regarding  these  cultures  reads:  "  Nothing  definite 
has  developed.  Two  of  the  tubes  of  ascitic  fluid  containing  rabbit's 
testicle,  covered  with  paraffin  oil,  have  developed  slight  cloudiness  as 
compared  with  the  corresponding  control  tubes,  but  microscopically  only 
fine  granules  can  be  detected  in  these  cultures.  Subcultures  on  agar 
remain  sterile.  Three  of  the  tubes  containing  ascitic  fluid  agar  with  the 
fragment  of  tissue  in  the  depth  have  remained  entirely  clear.  One  of 
these,  and  one  of  the  tubes  containing  the  rabbit's  kidney  have  become  con- 
taminated with  a  coarse  bacillus.     The  two  agar  cultures  remained  sterile." 

July  1.  The  patient  (Case  1)  was  brought  to  the  laboratory,  and  the 
areas  of  skin  upon  which  the  lesions  to  be  removed  for  study  were  situated 
were  scrubbed  with  soap  and  water,  then  with  alcohol  and  ether,  and  finally 
painted  with  iodin.  After  this  disinfection  the  papules  and  larger  nodules 
were  removed  with  sterile  forceps  and  scissors,  and  placed  in  sterile  Petri 
dishes.  At  the  same  time  as  the  removal  of  the  papules  a  number  of 
cultures  were  made  from  the  serum  and  blood  which  exuded  from  the  cut 
surface  of  the  tissue  after  removal  of  the  tumors.  In  all  about  ten  small 
papules  of  the  miliary  type,  and  of  reddish  brown  color,  measuring  about 
2  to  3  mm.  in  diameter,  were  removed.  Three  larger  nodular  areas, 
measuring  about  6,  7  and  8  mm.  in  diameter  were  also  removed  with 
scissors.  One  of  these  nodular  areas,  situated  over  the  anterior  surface 
of  the  right  foot,  and  measuring  about  6  mm.  in  diameter,  and  3  or  4  mm. 
in  height,  was  of  a  dark  reddish  color.  Upon  removal  it  was  found  in  the 
center  to  contain  a  necrotic,  cheesy,  greasy-like  material.  Cultures  and 
smears  were  made  from  this  material. 

Another  tumor,  measuring  about  7  mm.  in  diameter,  was  situated  below 
the  internal  malleolus  of  the  same  foot.  It  was  of  a  dark  reddish  color, 
the  surface  being  covered  by  a  blood  clot.  A  portion  of  this  tumor  had 
been  removed  by  us  on  June  20.  It  was  now  completely  removed  by  the 
scissors. 

The  third  tumor,  measuring  about  8  mm.  in  diameter,  was  situated 
midway  over  the  lower  third  of  the  anterior  surface  of  the  tibia.  The  skin 
over  it  was  a  little  darker  than  the  normal  color.  On  palpation  the  tumor 
seemed  a  little  soft  in  one  spot,  and  upon  removal,  one  area,  measuring 
about  4  mm.  in  diameter,  was  distinctly  softened  and  gelatinous.  Cul- 
tures were  made  from  some  of  this  gelatinous  material. 

The  fourth  tumor,  situated  over  the  internal  malleolus  of  the  left  foot, 
was  of  the  mulaire  type  and  measured  about  1.5  cm.  in  diameter  on  the 


EXPERIMENTS  91 

surface,  and  had  a  base  measuring  about  6  or  8  mm.  in  diameter  after 
removal.  The  surface  was  covered  with  a  moist  scab.  Cultures  were  also 
made  from  the  cut  surface  of  this  tumor  after  removal. 

Microscopical  Preparations.  —  Smears  were  made  from  the  cut  surface 
of  the  small  papules,  from  the  cut  surface  of  the  larger  nodules  (particu- 
larly the  cheesy  and  gelatinous  portions),  from  the  cut  surface  of  the 
tumor  partially  removed  on  June  20th,  and  from  the  cut  surface  of  the 
mulaire  nodule.  In  some  of  the  stained  preparations  from  the  papules 
a  few  yeast  cells  and  bacteria  were  encountered.  Those  from  the  cheesy 
and  gelantinous  material  showed  no  microorganisms.  In  the  preparations 
made  from  the  tumor  which  had  been  partially  removed  previously, 
numerous  yeast  cells  and  a  few  bacteria  were  present,  and  in  those  from 
the  mulaire  lesion  numerous  yeast  cells  and  very  large  numbers  of  cocci 
and  a  few  bacilli  were  present.  No  spirochaetae  were  discovered  in  any 
of  the  preparations. 

Cultures.  —  Thirty-six  cultures  were  made  with  fragments  of  the  lesions 
placed  in  ascitic  fluid  containing  pieces  of  rabbit's  testis  or  rabbit's  kid- 
ney; in  ascitic  fluid  agar  containing  rabbit's  testis  or  kidney;  in  ascitic 
agar  alone,  on  blood-serum,  egg  media  and  alkaline  agar. 

Some  of  the  cultures  were  made  by  planting  the  miliary  tumors  deep 
into  the  culture  mecUa.  Others  were  made  with  the  gelatinous  material 
scraped  from  the  cut  surface  of  one  of  the  tumors;  others  from  the  cheesy 
material  found  in  one  of  the  tumors;  and  still  others  with  small  bits  of 
tissue  cut  from  the  interior  of  the  nodular  tumor. 

Other  cultures  were  also  made  from  the  cut  surface  of  the  mulaire 
tumor.  Half  of  the  cultures  were  then  covered  with  paraffin  oil,  and  some 
placed  under  even  more  strictly  anaerobic  conditions  in  a  sealed  jar  con- 
taining phosphorus.  The  cultures,  together  with  a  larger  number  of 
corresponding  controls,  were  then  placed  in  the  incubator  at  thirty-seven 
degrees  centigrade. 

Cultures  on  agar  from  the  mulaire  lesion  at  its  base,  after  removal, 
showed  after  twenty-four  hours,  in  one  tube  thirty-six  colonies  of  a  white 
porcelain-like  appearance.  Microscopically  the  organism  was  a  coccus  and 
showed  great  variation  in  size  (Staphylococcus  alhus).  The  other  cultures 
inoculated  from  the  mulaire  lesion  after  three  weeks  in  the  incubator  also 
showed  this  organism  and  some  yeast  cells.  Some  of  the  cultures  from  the 
other  lesions  after  three  weeks  in  the  incubator  also  showed  bacterial  growth 
(usually  of  a  coccus,  or  in  a  few  instances  of  bacilli),  but  these  organisms 
were  not  constantly  present  in  the  inoculated  tubes  comprising  the  different 
.series,  and  from  their  characteristics  they  evidently  were  not  concerned  in 
the  primary  etiology  of  the  lesions.  In  the  ease  of  culture  No.  6,  made  in 
ascitic  agar  in  which  a  portion  of  a  verruga  papule  measuring  3  to  4  mm, 
was  placed  in  the  center  and  covered  with  paraffin  oil,  the  media  remained 


92  VERRUGA  PERUVIANA 

clear  for  about  two  weeks.  Subsequently  along  the  track  the  tissue  had 
taken  during  the  inoculation  of  the  media  a  distinct  haziness  appeared. 
The  tube  was  cut  and  microscopical  preparations  and  subcultures  made  in 
ascitic  agar  and  on  alkaline  agar.  No  spirochaetae  or  other  microor- 
ganisms could  be  demonstrated  in  the  microscopical  preparations  made 
from  the  culture,  and  apparently  no  growth  occurred  in  the  subcultures. 
In  culture  No.  24,  consisting  of  ascitic  agar  +  rabbit's  kidney  +  a  bit  of 
verruga  nodule  +  paraffin  oil,  slight  but  distinct  cloudiness  also  developed 
about  the  tissue  and  along  the  line  of  inoculation.  Microscopical  spec- 
imens stained  with  methylene  blue  and  Giemsa's  solution  showed  no  vis- 
ible microorganism,  only  granules.  A  subculture  in  alkaline  agar  gave  no 
growth.  No  growth  occurred  in  the  cultures  made  upon  blood-serum  or 
egg  media,  or  upon  the  agar  tubes  except  those  from  the  mulaire  lesion. 
Of  the  fifteen  cultures  sealed  and  placed  in  the  anaerobic  jar  with  phos- 
phorus, only  the  ones  made  from  the  mulaire  lesion  developed  any  growth 
when  subcultures  were  made  from  them.  Also,  there  were  no  appearances 
suggesting  growth  in  the  great  majority  of  the  cultures.  Fresh  and  stained 
microscopical  preparations,  however,  were  made  from  them  all,  for  the  last 
time  three  weeks  after  their  inoculation.  No  definite  microorganisms  were 
detected,  though  numerous  granules  and  the  remains  of  cells  were  present 
in  all.  In  cultures  Nos.  27  and  28,  containing  ascitic  fluid  -|-  rabbit's  tes- 
tis +  a  fragment  of  verruga  nodule  (no  paraffin  oil),  slight  cloudiness 
gradually  developed.  Stained  microscopical  preparations,  methylene  blue 
and  Giemsa's  solution  being  employed,  showed  no  microorganisms,  only 
a  large  number  of  deeply  staining  granules.  Subcultures  on  agar 
remained  sterile.  A  portion  of  these  cultures  when  they  were  one  month 
old  was  inoculated  into  Rabbits  Nos.  61  and  62  (q.  v.)  with  a  negative 
result. 

Inoculation  of  Animals.  —  A  suspension  of  six  of  the  small  papules,  and 
of  two  of  the  larger  tumors,  measuring  about  6  to  7  mm.  in  diameter,  were 
ground  up  in  8  c.c.  of  saline  solution.  2.5  c.c.  of  this  suspension  were  then 
filtered  through  a  Berkefeld  filter.  The  remaining  5.5  c.c.  of  the  suspen- 
sion of  the  tumors  were  then  injected  into  the  testes  of  five  rabbits  (see 
Rabbits  Nos.  8  to  12),  about  an  equal  amount  of  fluid  being  injected  into 
each  animal,  and  both  testes  being  inoculated  in  all  cases.  The  filtrate 
had  been  diluted  with  12  c.c.  of  saline  solution.  1  c.c.  of  this  solution  was 
then  injected  into  both  testes  of  two  rabbits.  From  the  central  and  inner 
portion  of  the  mulaire  tumor  a  small  bit  of  tissue  measuring  about  6  mm. 
in  diameter  was  removed  and  ground  up  in  2  c.c.  of  saline  solution.  One 
c.c.  of  this  suspension  was  then  injected  into  each  testis  of  a  rabbit. 

Two  other  rabbits  were  inoculated  as  follows:  One  received  an  intra- 
peritoneal inoculation  with  the  gelatinous  material  remaining  from  the 
suspension  used  in  the  inoculation  of  Rabbits  Nos.  8  to  12.     Two  trocar 


EXPERIMENTS  93 

fulls  were  introduced.     The  second  was  injected  intraperitoneally  with 
about  10  e.c.  of  the  filtrate  of  the  suspension  spoken  of  above. 
These  inoculations  may  be  summarized  as  follows : 

Testis  inoculation  with  suspension,  of  Rabbits  Nos.  8,  9,  10,  11  and  12. 
Testis  inoculated  with  filtrate,  Nos.  13  and  14. 
Testis  inoculated  with  verruga,  mulaire  type,  No.  15. 
Intraperitoneal  inoculation  \\ath  verruga  suspension,  No.  16. 
Intraperitoneal  inoculation  with  verruga  su-spension  filtrate.  No.  17. 

Dark-Field  Microscope.  —  Fresh  preparations  were  made  from  the  serum 
and  blood  from  the  cut  surface  of  the  miliary  and  nodular  tumors,  and  from 
the  cheesy  and  gelatinous  material.  No  spirochaetae  or  phenomena  of 
importance  were  observed. 

Preservation  of  Material.  —  The  following  material  was  preserved  for  fu- 
ture histological  study:  (1)  a  slice  of  the  mulaire  lesion  in  Zenker's  solution; 
(2)  slices  of  the  mulaire  lesion,  and  one  small  slice  of  the  nodular  lesion 
previously  excised,  in  corrosive  alcohol,  and  one  slice  of  the  mulaire  and  one 
bit  of  the  nodular  lesion  previously  excised,  in  ten  per  cent  formalin. 

Inoculation  of  Animals 

Rabbit  No.  8.  —  Testes  inoculation  with  suspension  of  verruga. 
July  1,  1913.     Inoculated  both  testes  with  0.5  c.c.  of  a  suspension  of  freshly  re- 
moved small  verrugas  and  gelatinous  material  from  pre\aously  operated  lesion, 
ground  in  salt  solution. 

July    3.     10.15  A.M.  Temp.  102°.6  F.  Both  testes  ill-defined  as  though  rup- 
tured.     Right  side  of  scrotum,  subcu- 
taneous  oedema   general.       Left   side, 
anterior  part  oedematous. 
5.      8.25    "  "        99°.9.      Testes  movable  wathin  scrotum.  Oedema 

of  both  less  tense,  but  more  induration. 
Uneven  on  left. 
"       7.       9.30    "  "        99°.9.       Scrotum    normal.     Left    testis,    small 

nodules    scattered    through    substance. 
Right,  firm  nodule  in  anterior  portion. 
"       9.     10.40    "  "       100°.6.      Right,  definite  nodule  i  distance  from 

anterior  end.      Left  testis,  barely  per- 
ceptible, uneven  granules  in  substance. 
"      11.     11.15    "      (not  taken)  Right  te-stis,  definite  nodule  central  por- 

tion, firm,  several  mm.  in  diameter. 
"      13.      4.05  p.m.  Left,  granular;   right,  fairly  distinct  firm 

nodule. 
"      15.     10.16  a.m.  Left,    shghtly    granular;     right,   fairly 

distinct  firm  nodule,  rather  soft,  flabby. 
18.     11.20    "  Both  uneven  in  consistence.     No  defi- 

nite nodule.     Soft  and  flabby. 
20.     10.42    "  Testes  uneven  consistence,  small,  atro- 

phied. 
"     26.     11.55    "  Testes  negative,  soft,  uneven. 


5. 

8.33    " 

7. 

10.00    " 

9. 

10.45    « 

11. 

11.20    " 

13. 

4.10  P.M. 

15. 

10.17  A.M. 

94  VERRUGA  PERUVIANA 

July  27.  Killed.  Both  testes  show  2  to  3  orange  yellow  lesions,  of  dry  con- 
sistence, irregular  in  outline,  2  mm.  across;  each  shows  two  lesions,  otherwise 
atrophied.  Spleen  rounded,  slightly  enlarged.  Whitish  scar  ventral  edge  liver. 
Emaciated,  losing  fur.     Tissue  in  Zenker's  solution. 

Rabbit  No.  9.  —  Testes  inoculation  with  verruga  suspension. 
July    1,  1913.     Inoculated  both  testes  with  0.5  c.c.  of  a  suspension  of  freshly 
removed  small  verrugas,  ground  in  salt  solution. 

July    3.     10.17  A.M.  Temp.     99°.9  F.  Testes    free    in    scrotum;    tender,   soft 

throughout. 
'       101°.7.      Testes  free,  soft,  no  lesion. 
'       100°.3.  "  "         "     homogeneous,     slight 

unevenness  left  side. 
'         99°. 9.       Right  testis  normal;   left  normal. 
Both  testes  normal. 
Testes  slightly  more  tense.     No  definite 
nodules. 

Right  testis   no   definite  lesion,   tense. 
Left,  shows  a  definite  firm  mass  in  cen- 
tral portion  in  addition. 
"      18.     10.20    "  Right  testis,  indefinite  nodule  or  indura- 

tion anterior  to  center.     Left,  definite 
nodule  apparently  large. 
"     21.     11.50    "  Barely   perceptible   nodule   center   left 

testis. 
"     22.      4.00  P.M.  Chloroformed  and  left  testis  removed 

with  only  moderate  aseptic  precautions 
(alcohol) .  Right  also  now  shows  minute 
palpable  nodule  now  that  animal  is  tied. 
On  section  left  shows  pale  yellowish 
lesion,  dry,  firm,  bulging  from  surface;  ^ 
placed  in  Zenker's  solution.  Other  for 
inoculation  of  Rabbit  No.  48. 
"     26.     11.55    "  Left  side  of  scrotum  somewhat  swollen. 

Right  testis,  a  definite  firm  nodule  in 
central  portion. 
August  3.  Left  scrotum,  no  distinct  nodule.  Killed.  Right  testis  removed. 
This  shows  areas  2X3  mm.  sUghtly  more  opaque  and  sUghtly  yellower  than  testis, 
but  with  distinctly  defined  broader  necrotic  area.  Spleen  small;  scar  on  edge  of 
liver.  Small  amount  of  fluid  and  gelatinous  clot  in  peritoneal  cavity.  Tissue 
in  Zenker's  solution. 

Rabbit  No.  10.  —  Testes  inoculated  with  verruga  suspension. 
July  1,  1913.     Inoculated  both  testes  with  0.5  c.c.  of  a  suspension  of  verruga 
material  as  in  Rabbits  Nos.  8  and  9. 

July    3.     10.20  A.M.  Temp.     99°.4  F.  Testes  movable  within  scrotum.     Slight 

increase     in     tension.      Homogeneous 
throughout. 
"       101°.  1.       Testes  movable,  soft  throughout. 

101°.  1.  "  "  "    flabby,     homoge- 

neous. 
"       9.     10.45    "  "       101°.  Right    scrotum    shghtly    oedematous; 

left  testis  normal. 


3. 

10.20  A.M, 

5. 

9.41    " 

7. 

10.05    " 

July  11. 

11.20  A.M, 

"   13. 

4.10  P.M. 

«   15. 

10.20  A.M. 

«   18. 

10.25  " 

«   21. 

10.50  « 

"  22. 

4.30  P.M. 

EXPERIMENTS  95 

Temp.  Both  testes  normal. 

Slight    induration    anterior    pole    left. 
Right  testis  negative. 
Neither   testis   shows   definite   nodule. 
Left,    slightly    more    tense.       Neither 
flabby. 

Neither  shows  a  definite  nodule.  Tense. 
Barely  perceptible  thickening  in  center 
of  the  left.  Questional:)le  in  center  of 
right. 

Left  testis  excised. 
Testis   shows   a   pale   slightly   opaque 
lesion  1  to  1.5  mm.  across,  but  extending 
longitudinally  5  mm.  or  more.     A  por- 
tion used  to  inoculate  Rabbit  No.  49. 
"     26.     11.59  A.M.  Left  side  of  scrotum  swollen.      Right 

testis  a  minute  firm  nodule. 
"     30.  Dry  crust  left;    no  distinct  nodule  in 

right. 
August  3.     Killed.     Left  side  of  scrotum  healed.     Right  side  barely  perceptible 

thickening.     Right  testis  shows  one  lobule  more  opaque  and  yellower  than  normal. 

Small  scar  at  edge  of  Uver.     Spleen  small.     No  tissue  saved. 

Rabbit  No.  11. — Testes  inoculated  with  verruga  suspension. 
July  1,  1913.     Inoculated  both  testes  with  0.5  c.c.  of  a  suspension  of  verruga 
material  as  in  Rabbits  Nos.  8  and  9. 

July    3.     10.25  A.M.  Temp.  100°. 3  F.  Testes  freely  movable.     Homogeneous, 

soft. 
"       5.       8.50    "  "       100°. 4.      Testes  freely  movable.     Posterior  end 

left,  hard,  and  here  slight  swelling  of 
scrotum. 
7.     10.10    "  "       101°.4.       Right  testis  soft,  homogeneous.      Scro- 

tum normal.     Left  side  of  scrotum  swol- 
len throughout,  tense.     Testis  hard  and 
fixed. 
9.     10.50    "  "       104°.2.       Right  testis  normal.     Left  side  of  scro- 

tum swollen.     Testes  fixed. 
"      11.     11.25    "  Right  testis  normal.     Left  testis  firm, 

swollen,  with  epididymis  forming  firm 
mass. 
"      13.       4.15  P.M.  Right    not    found.     Left    testis    fixed. 

Swelling  less. 
"      15.     10.34  A.M.  Right  testis  rather  tense.     No  definite 

nodules.    Left,  fixed,  indurated  through- 
out. 
"      18.  No  definite  nodule.     Inflammation,  left, 

subsiding,  leaving  posterior  end  firm. 
"     21.  Right  firm.     Definite  nodule  in  anterior 

portion.     Left,  hard  posteriorly. 
"      26.  Right,  anterior  indurated.     Left,  poste- 

rior portion  inchirated. 
July  27.     Killed.     Right  testis  presents  a  group  of  Ic^sions,  ^  used  to  inoculate 
Rabbit  No.  56.     Other  testis  shows  a  large  yellow  nodule  7X5  mm.,  on  section 


96  VERRUGA  PERUVIANA 

cheesy  and  soft.     Smears  fresh  and  stained,  and  cultures  on  agar  negative  for 
microorganisms.     Other  organs  negative.     Tissue  in  Zenker's  solution. 

Rabbit  No.  12.  —  Testes  inoculation  with  verruga  suspension. 
July  1,  1913.     Inoculated  both  testes  with  0.5  c.c.  of  a  suspension  of  verruga 
material  as  in  Rabbits  Nos.  8  and  9. 

July    3.     10.30  A.M.  Temp.  101°.6  F.  Oedema  both  sides  of  scrotum.     Punc- 
ture of  skin  on  right. 
"       5.      8.45    "  "       100°.         SweUing  of  right  side  of  scrotum;  skin 

puckered.     Testis  rather  hard  but  slips 
through    scrotum.       Left     testis     not 
found.     Left  scrotum  normal. 
"       7.     10.20    "  "         99°.  Right  testis  normal,  homogeneous.    Im- 

possible to  get  left  down. 
"       9.     10.55    "  "         99°. 6.       Shght  induration  at  anterior  and  poste- 

rior end  of  right  testis.     Left  normal. 
"     11.     11.30    "  Definite  firm  nodule  posterior  portion 

right  testis,  and  induration  at  anterior 
end.     Left  normal. 
"     13.      4.20  p.m.  Definite  firm  nodule  at  posterior  of  right. 

Left,  small,  soft. 
"     15.     10.30  A.M.  Firm  nodule  near  posterior  end  right. 

Both  flabby  and  soft. 
"     18.  No  definite  nodule.     Right,  uneven  con- 

sistence.    Both  flabby. 
"21.  No  definite  nodule. 

"     26.  Both  testes  uneven.     No  definite  nod- 

ules. 
July  27.     Killed.     Right  testis  shows  yellowish  nodules  with  grayish  periphery 
and  smaller  whitish  gray  areas  within.      Left,  shows  one  minute  grajdsh  area. 
Other  organs  negative.     Tissue  in  Zenker's  solution.     Fresh  and  stained  smears 
with  Wright  and  Giemsa  negative  for  microorganisms.     Cultures  on  agar  negative- 

Rabbit  No.  IS.  —  Testes  inoculation  with  verruga  filtrate.  Filtrate  obtained 
by  diluting  suspension  used  to  inoculate  Rabbits  Nos.  8  to  12  and  15,  16,  and  then 
passing  through  Berkefeld  filter. 

July  1,  1913.    Both  testes  inoculated  with  0.5  c.c.  of  filtrate. 
July    3.     10.10  a.m.  Temp.   100°.3F.  Testis   shghtly   harder   on   right   side. 

No  nodule  in  either. 
Testes,  no  lesion,  soft,  normal. 

It  ti  u  u  a 

Both  testes  normal. 

u  u  a 

"       SmaU. 
"  "  "       Right  slightly  more 

tense. 
"     18.  No  definite  nodules.    Right  much  larger 

than  left  and  more  tense. 
"     21.  No  definite  nodules.     Right  much  larger 

than  left  and  more  tense. 
'*     26.  No  definite  nodules. 

July  28,  1913.  Killed  by  chloroform.  The  ventral  edge  of  the  liver  shows 
whitish  scar-like  lesion  about  2  cm.  across.    Other  viscera  show  no  gross  lesions. 


5. 

8.38    " 

u 

100°. 

7. 

10.25    " 

u 

lor.i 

9. 

11.00    « 

« 

101°.7, 

11. 

11.30    " 

13. 

4.15  P.M. 

15. 

10.40  A.M. 

EXPERIMENTS  97 

Smaller  of  the  two  testes  appears  normal  throughout.  Larger  testis  shows  a  rather 
translucent  lobule  or  nodule  approximately  2  mm.  across,  only  differing  from  the 
normal  in  its  translucency.     Tissue  in  Zenker's  solution. 

Rabbit  No.  14-  —  Testes  inoculation  with  verruga  filtrate. 

July  1, 1913.  Both  testes  inoculated  with  0.5  c.c.  of  a  filtrate  obtained  by  dilut- 
ing suspension  used  to  inoculate  Rabbits  Nos.  8  to  12  and  15,  16  with  4  X  volume 
of  salt  solution,  and  then  passing  through  Berkefeld  filter  which  it  did  quickly. 

July    3.     10.07  A.M.  Temp.     99°.8  F.  Testes  soft  and  normal. 


a. 

7. 

10.27    " 

99° 

.u. 

.9. 

"      "       flabby. 

9. 

10.55    " 

«       100° 

.4. 

Both  testes  normal. 

11. 

11.30    « 

11                a                  u 

13. 

4.25  P.M. 

U                       l(                           it 

15. 

9.42  A.M. 

"       Not  flabby. 

18. 

No  nodules,  soft,  flabby. 

21. 

«          11             11         11 

26. 

11          11             11         11 

"     30.  Neither  testis  shows  any  nodule. 

August  9.     Killed.     Both  testes  normal.     Smear  and  cultures  made.     Cysticer- 
cus  in  omentum.     No  tissue  saved. 

Rabbit  No.  15.  —  Testes  inoculation  with  verruga  mulaire  type. 
July  1,  1913.     Both  testes  inoculated  with  0.5  c.c.  of  a  suspension  of  material 
from  interior  of  large  mulaire  lesion. 

July    3.     10.03  A.M.  Temp.  102°.5  F.  Testes  swollen,  tender,  red,  tense  and 

firm. 
5.       8.53    "  "       102°.3.     Testes  slightly  movable;    not  felt  on 

account  of  swelling  of  scrotum  which  is 
tense,    brawny    and    dusky    purple    at 
posterior  ends  of  both. 
7,     10.30    "  "       101°.  Right   side   of   scrotum,    skin   normal. 

Testes  and  epididymis,  dark  red  blotches 
posterior.      Left  side  of  scrotum  still 
tense,  but  swelling  less.     Testes  fixed, 
hard. 
9.     11.02    «  «       102°.6.       Right  testis,  shghtly  discolored,  purplish 

and    of    slightly    uneven    consistence. 
Swelling  has  subsided  in  left  side  of  scro- 
tum.    Still  some  induration. 
"      11.     11.35    "  Right  testis,  small,  hard,  uneven,  shps 

readily.      Left  side  of  scrotum  fairly 
firm  nodule  with  opaque  yellowish  ma- 
terial beneath  skin. 
"      13.       4.30  P.M.  Right,  homogeneous.     Left,  drawn  up, 

firm  cord  felt. 
"      15.     10.45  A.M.  Right  uneven  consistence.     Left,  palpa- 

ble, firm,  tender. 
"      18.  Both  indurated,  uneven.     Scrotum  un- 

even, indurated. 
"      19.  No  definite  nodule  except  abscess  at  tip 

of  left  scrotum. 
"     26.  No  definite  nodule.     Uneven  nodules  in 

epididymis  of  the  left. 


98  VERRUGA  PERUVIANA 

July  29.  Killed.  Testes  both  atrophied,  and  each  shows  several  golden  almost 
orange  yellow  areas  definitely  circumscribed  but  rather  of  more  irregular  form 
than  rounded,  and  surface  overlying  is  depressed  as  though  from  loss  of  substance. 
On  section  a  quantity  of  whitish  thick  pus  expressed  from  one  side  of  the  lesion. 
In  addition  there  is  a  translucent  nodule,  not  yellow,  possibly  the  result  of  atrophy 
of  the  remainder  of  the  testis.  Other  organs  present  no  lesion.  Microscopical 
preparation  from  the  lesion  in  the  testis  shows  numerous  polymorphonuclear 
leukocytes  and  fair  number  of  cocci.  Culture  on  agar  gives  a  rich  growth  of  the 
same  organism.     Tissues  in  Zenker's  solution. 

Rabbit  No.  16.  — Intraperitoneal  inoculation  with  verruga  suspension. 
July  1,  1913.     Introduced  greater  part  (2  trocars  fuU)  of  gelatinous  material 
remaining  from  suspension  into  peritoneal  cavity. 

July    3.     10.00  A.M.  Temp.  103°. 6  F.  Thickening  at  point  of  inoculation  be- 
neath skin. 

Implant  soft;  not  markedly  inflamed. 

Implant  soft;  not  markedly  inflamed. 

Subcutaneous  mass  not  increased  in  size. 

Two  small  accessory  areas  near  large 

implant. 

Subcutaneous    main    implant    smaller; 

more  elevated;  subsidiary  larger. 

Implants  distinctly  smaller,  rounder. 

Implants  slightly  smaller. 
"     26.  Subcutaneous  implants  smaller. 

July  29.  Killed.  The  implant  beneath  the  skin  is  found  to  consist  of  a  sac 
of  dark  grayish  caseous  material  mingled  with  whiter  material.  There  is  also  a 
small  elongated  mass  embedded  in  the  muscle,  and  extending  through  the  abdomi- 
nal wall.  Within,  there  is  a  lesion  2  mm.  across,  somewhat  flattened,  attached 
to  the  previously  mentioned  area  by  a  long  cord  containing  blood-vessels,  attached 
to  the  cecum  by  a  cord  2.5  cm.  in  length,  and  an  area  measuring  9X6X5  mm. 
This  is  of  duU  brownish  color  at  one  extremity,  surrounded  by  a  bright  red  zone. 
The  remainder  of  the  area  is  mottled  pink,  whitish  and  dark  gray.  In  the  cord 
by  which  this  is  attached  is  a  small  red  area  less  than  1  mm.  across.  The  larger 
one  on  section  is  found  to  have  a  dry  surface,  and  is  apparently  for  the  most  part 
necrotic.  On  the  left  lobe  of  the  hver,  near  the  border,  is  a  lesion  measuring 
5  mm.,  rounded  and  somewhat  grayish  in  color,  but  translucent.  On  section  this 
is  found  to  be  somewhat  cystic,  and  contains  grayish  material  mingled  with  dark 
red  blood.  The  spleen  shows  one  minute  translucent  area  about  midway  on  its 
lateral  border.  The  peritoneum  is  otherwise  smooth  and  shows  no  lesions.  Cul- 
tures from  the  cut  surface  of  the  lesions  remained  sterile. 

Rabbit  No.  17.  —  Intraperitoneal  inoculation  with  verruga  filtrate. 

July  1,  1913.  Injected  about  10  c.c.  of  filtrate  of  suspension  of  ground-up 
verruga  material  (used  to  inoculate  Rabbits  Nos.  8  to  12  and  15, 16)  into  peritoneum. 
This  suspension  was  diluted  with  four  times  its  volume  of  salt  solution,  and  it  passed 
quickly  through  the  Berkefeld  filter,  giving  a  clear  slightly  pink  fluid. 

July 


5. 

8.58 

a 

u 

101°.9. 

7. 

10.35 

11 

a 

101°.9. 

9. 

11.09 

u 

u 

102°.3. 

11. 

11.40 

ti 

15. 

10.45 

li 

u 

101°. 

18. 

11.35 

11 

u 

100°.8. 

21. 

11.07 

u 

u 

102°.8. 

3. 

9.30  A.M.- 

Temp.  100°.5r. 

5. 

8.56    « 

«       101°.5. 

7. 

10.37    " 

"       100°.8. 

9. 

11.05    « 

"       101°.6. 

11. 

11.40    " 

No  observation. 

EXPERIMENTS  99 

July   15.     10.47  A.M.     Temp.  99°. 8. F.  Blood  smear  stained  by  Giemsa,  nega- 
tive. 
"      18.     10.08    "  "       102°. 

July  29.     Klilled.     Peritoneum  and  aU  organs  appear  normal.     No  tissue  saved. 

Rabbit  No.  28.  —  Testes  inoculation  2d  transfer  (trocar). 

July  7,  1913.     Both  testes  inoculated  by  trocar  with  bits  of  lesion  of  Rabbit 
No.  2  about  one  hour  after  kiUing  latter. 

July    9.     11.35  A.M.  Temp.  102°.4  F.  Subcutaneous  white  nodule  in  right  side 

of  scrotum  with  reddening  at  periphery. 
Left  testis,  sUght  induration  anterior 
portion. 

"      11.     12.00  M.  White  nodule,  subcutaneous,  ventral  as- 

pect right  side  of  scrotum  2X3  mm. 
No  palpable  nodule  in  interior.  Left 
scrotum  shows  bluish  discoloration  few 
mm.  in  diameter  with  ill-defined  border. 
Slight  induration  anterior  to  central 
portion. 

"      13.      4.45  P.M.  Testes  rather  tense.     No  nodule  in  in- 

terior, soft. 

"      15.     11.23  A.M.  Right  not  expressed.     SUght  swelling  of 

posterior  portion  of  scrotum.  No  defi- 
nite nodule  in  interior. 

"      18.     10.45    "  Small  nodules  at  surface  of  right,  whitish 

in  center,  bright  red  at  periphery.  Left 
large,  tense.  No  definite  nodule.  Point 
of  puncture  reddened  and  thickened. 

"      19.  Spot  not  so  bright  red  in  right  testis. 

The  left  shows  a  large  swelling  in  poste- 
rior portion. 

"     21.  Spot  whitish  in  right  testis;  no  nodule. 

Marked  oedema  of  left  scrotum. 

"     26.  Spot,  right     scrotum,    extended,   more 

diffuse.  Left,  harder  posteriorly.  No 
definite  nodule  in  either. 

"      29.  Both    practically    negative.     Soft. 

«     30. 
August  8.      Right  testis,  palpable  nodule,  small.     Killed.     T^eft  testis,  an  ill- 
defined  area  on  section  with  dark  gray  markings.     Right,  less  well  defined  small 
nodule.     Testes  in  Zenker's  solution. 

Rabbit  No.  29.  —  Testes  inoculation,  2d  transfer  (trocar). 

July  7,  1913.     Both  testes  inoculated  by  trocar  with  bits  of  lesion  of  Rabbit 
No.  2  about  one  hour  after  killing  latter. 

July    9.     11.35  A.M.  Temp.  102°.  1  F.  Bluish  spot  posterior  end  of  right  testis. 

Barely  perceptible  nodule  anterior  por- 
tion.    Left,  induration  at  anterior  por- 
tion. 
"      11.     12.05  P.M.  Right  no  definite  nodule.      Left,  shght 

induration  in  central  portion  of  testis. 
"      13.      4. .50    "  Left  tense,  indurated  in  center.     Right 

tense,  slight  swelling. 


100  VERRUGA  PERUVIANA 

No  definite  nodule  in  either,  tense. 


July  15. 

11.20  A.M 

«     18. 

10.50    " 

«     19. 

3.30  P.M. 

«     21. 

«     26. 

12.30    " 

"     29. 

"     30. 

August  8. 

KiUed. 

Neither  testis  shows  any  definite  mass. 
Left  testis  area  of  dark  discoloration  without  reaction  of 
surrounding  tissue.  Right  shows  area  of  shghtly  denser  tissue  with  some  dis- 
coloration. No  weU-defiaed  border  or  bulging.  Other  organs  normal.  Tissue 
in  Zenker's  solution. 

Rabbit  No.  30.  ■ — •  Testes  inoculation,  2d  transfer  (syringe). 
July  7,  1913.     Both  testes  inoculated  with  0.5  c.c.  of  suspension  of  lesion  of 
Rabbit  No.  2  ground  in  salt  solution,  one  hour  after  killing. 

July    9.     11.40  a.m.  Temp.  103°.3  F.  Both  testes  homogeneous. 

"      11.     12.05  P.M.  "  "      normal. 

"      13.       5.00    " 

"      15.     11.25  A.M.  "  "  "       rather  tense. 

"      18.     10.52    "  Tense,  large.     No  palpable  nodule. 

«     19.      3.20  p.m. 

"     26.     12.31    "  "  "  "  "  " 

it        OQ  "  "  «  "  " 

«       on  u  u  a  u  u 

August  8.  Killed.  Both  testes  show  iU-defined  area  with  some  dark  pig- 
mentation.    Organs  normal.     No  culture.     Tissue  in  Zenker's  solution. 

Rabbit  No.  31 .  —  Testes  inoculation,  2d  transfer  (syringe) . 
July  7,  1913.     Both  testes  inoculated  with  0.5  c.c.  of  suspension  of  lesion  of 
Rabbit  No.  2  ground  in  salt  solution  one  hour  after  removal. 

July    9,    11.42  a.m.  Temp.  101°.5  F.     Slight  thickening  of  skin  at  point  of 

inoculation.     Testis  homogeneous.    No 
testis  found  on  left  side. 
"      11.     12.10  p.m.  Right  testis  normal.     Left  not  found. 

«     15.     11.33  a.m. 

"     18.     10.55    "  «  "     tense.    Left  not  found. 

"     19.      3.25  P.M.  "  "          "  "       "        " 

"     21.  A.M.  "  "     no  nodules. 

"     26.     12.34  p.m. 

"     29.  Definite  nodule  in  right  testis. 

Aug.    8.  "        large  nodule  in  right  testis. 

August  9.  Killed.  Right  testis  shows  six  minute  elevations  on  surface  mem- 
brane. Within  its  substance  is  a  rounded  nodule  measuring  about  5  mm.  in 
diameter,  defuiitely  circumscribed,  whitish  in  color.  On  section  there  appears 
to  be  no  necrosis.  Left  is  small,  atrophied.  Other  organs  appear  normal.  Micro- 
scopical preparations  show  no  microorganisms.  Two  cultures  on  agar  from  the 
lesion  remain  sterile.  Lesion  ground  in  saline  solution  and  portion  of  suspension 
inoculated  into  Rabbits  Nos.  74-77  (q.v.). 

Rabbit  No.  32. —  Intravenous  inoculation,  2d  transfer. 
July  7,  1913.     Two  c.c.  of  suspension  injected  into  ear  vein. 
July    9.     11.40  a.m.    Temp.  102°.4F. 
"     11.     12.10  p.m.         "       102°.5. 


EXPERIMENTS  101 

July  15.  11.26  a.m.     Temp.  102°.  F.  Blood  (Giemsa)  negative. 

"      18.  11.57    "  "       101°.6. 

August  8.  Killed.     All  organs  appear  normal.     Testes  very  small.    No  tis- 
sue saved. 

Rabbit  No.  44-  —  Testes  inoculation,  2d  transfer  (syringe). 
July  14,  1913.     Both  testes  inoculated  with  0.5  c.c.  of  suspension  made  by  grind- 
ing ^  smaller  lesion  of  Rabbit  No.  3  in  small  amount  of  salt  solution.     Inoculation 
soon  after  kilUng  of  Rabbit  No.  3. 

Both  testes  normal. 

Rather  small,  firm. 


July  15. 

12.08  P.M. 

«     18. 

12.16     " 

«     21. 

12.02     " 

«     26. 

12.47     « 

"     29.  Appears  to  be  slight  local  sweUing.     No  definite  nodules. 

Aug.    3.  Left  testis  shows  a  definite  nodule.     Right  none. 

August  8.  No  nodule.  Killed.  Both  testes  (left  larger)  show  rather  ill- 
defined  shghtly  yellowish  lesions  1-3  mm.  Testes  in  Zenker's  solution.  Other 
organs  normal. 

Rabbit  No.  45.  —  Testes  inoculation,  2d  transfer  (syringe). 
July  14,  1913.     Testes  inoculation  with  0.1  c.c.  of  suspension  of  ^  of  smaller 
lesion  of  Rabbit  No.  3  in  salt  solution  in  order  to  observe  effect  of  small  dose. 
July  15.     12.08  P.M.     Testes  normal.     Right  small  and  soft. 


"  "       both  normal. 

Right  atrophied.     Left  large  full.     No  nodule. 

((  U  «  <(  U  ((  (( 

ed.     Both  testes  show  a  small  yellowish  nodule.     Testes  in 
Other  organs  normal.     . 

Rabbit  No.  46.  —  Testes  inoculation,  2d  transfer  (syringe). 
July  14,  1913.     Both  testes  inoculated  with  0.5  c.c.  of  suspension  of  larger  lesion 
of  Rabbit  No.  3  in  salt  solution.     This  was  done  nearly  three  hours  after  removal. 
July  15.     12.06  P.M.     Soft;  oedema  scrotum. 

Testes  tense,  normal.     No  oedema  of  scrotum. 


u 

18. 

12.15 

u 

21. 

12.02 

a 

26. 

12.45 

u 

29. 

Aug. 

,    3. 

Augi 

List  8. 

Kil 

oker' 

s  solution. 

15. 

12.06  P.M. 

18. 

12.14    " 

21. 

12           M. 

26. 

12.40  P.M. 

29. 

"  both  show  definite  firm  nodule,  readily  palpable. 
Left  testis  excised,  cord  tied  with  silk,  and  scrotum  left 
open.  Testis  shows  nodule  near  anterior  end  causing  sur- 
face to  bulge.  On  section  nodule  whitish  more  opaque 
than  testicular  tissue,  3-4  mm.  No  yellow  coloration. 
Microscopical  preparation  and  culture  made  from  lesion; 
and  suspension  of  lesion  inoculated  into  Rabbits  Nos.  57, 
58,  59  and  60.  No  tissue  preserved.  The  microscop- 
ical i)reparation  shows  no  microorganisms.  The  culture 
remained  sterile. 

Aug.    3.  Left  healed  clean.    Slight  oedema.     Right  large  nodule. 

Scrotum  slightly  oedematous. 

Sept.  16.  Scrotum  somewhat  thickened  and  possibly  oedematous 

over  central  portion  of  testis.  A  small,  firm  mass  is  pal- 
pable within  testis. 


102  VERRUGA  PERUVIANA 

Sept.  19.  Killed.  The  right  testis  presents  a  nodule  of  tissue  of  firm  consistence 
measuring  6  X  3.5  mm.,  which  on  section  bulges  from  cut  surface  and  is  slightly 
less  translucent  than  the  normal  surrounding  tissue  and  has  a  yellowish  tint.  It 
appears  homogeneous  throughout  and  there  are  no  areas  of  necrosis.  The  other 
organs  show  no  lesions  of  importance.  Animal  appears  to  have  been  in  good  health. 
The  smaller  half  of  the  testis  lesion  placed  in  Zenker's  solution.  The  remainder 
is  used  for  inoculation  of  two  rabbits  Nos.  90  and  91  (Table  i,  page  122). 

Dog  No.  2.  —  Testes  inoculation. 

July  16,  1913.  Both  testes  inoculated  with  0.5  c.c.  of  a  suspension  of  subcu- 
taneous nodule  from  Dog  No.  1  ground  in  salt  solution. 

July  18.     12.20  P.M.     Scrotum  appears  normal.     Nothing  over  thorax. 
«     21.  Nothing  abnormal  noted. 

«     26.  Testis  firm,  movable.     SHght  excoriation  of  scrotum. 

«     29.  Negative. 

Aug.    3.  Right  testis,  uneven  surface. 

August  8.  Killed  by  chloroform.  On  the  right  testis  is  a  small,  firm,  reddish 
nodule.  On  section  the  anterior  end  shows  dark  discoloration  with  considerable 
reddening.  This  portion  is  apparently  necrotic  and  more  opaque  than  normal. 
Left  testis  shows  a  reddish  firm  nodule  about  8  mm.  across  within  its  substance. 
It  bulges  shghtly  from  the  cut  surface.  Tissue  preserved  in  Zenker's  &  Kaiserling's 
solutions  after  photograph  was  taken. 

Bog  No.  3.  —  Male.     Subcutaneous  and  intraperitoneal  inoculation. 
July  16,  1913.    Injected  with  1  c.c.  of  same  suspension  as  used  in  Dog  No.  2 
subcutaneously  over  right  costal  cartilages  near  free  border.     1  c.c.  of  same  material 
injected  intraperitoneally  to  right  of  median  fine  a  few  cm.  from  the  inguinal  line. 
July  18.     12.21  P.M.     Appears  normal. 
"     21.  Nothing  abnormal. 

«     26.  Nothing  abnormal.    No  subcutaneous  oedema. 

"     29.  Negative. 

Aug.    3.  Negative. 

August  8.  Killed  by  chloroform.  Near  costal  margin  is  a  small  red  nodule, 
measuring  about  3  mm.,  embedded  in  the  subcutaneous  fat.  The  peritoneal  surface 
is  normal.  On  incision  the  subcutaneous  lesion  is  found  to  contain  a  large  amount 
of  blood. 

Rabbit  No.  47.  —  Testes  inoculation. 

July  16,  1913.  Each  testis  received  0.5  c.c.  of  suspension  subcutaneous  nodule 
from  Dog  No.  1. 

July  18.     12.24  p.m.     Testes  full,  normal. 
"     26.      1.07    "       Barely  perceptible  nodule  in  both  testes;  slightly  larger 

in  right. 
«     29.  Definite  small  nodule  in  both. 

Aug.    3.  Definite  large        "        "      " 

August  30.  Found  dead.  Small  yellowish  area  found  in  each  testis.  Other 
organs  normal.     Tissue  in  Zenker's  solution. 

Rabbit  No.  54.  —  Testes  inoculation,  2d  transfer  (sjo-inge). 

July  22,  1913.  Inoculated  left  testis  with  0.5  c.c;  right  with  0.25  c.c.  of  a 
suspension  of  nodule  of  left  testis  Rabbit  No.  9,  ground  in  salt  solution,  and  used 
within  twenty  minutes. 

July  26.  1.10  P.M.  Neither  testis  shows  a  definite  nodule.  Left  considera- 
bly larger  and  fuUer. 

Aug.    3.  No  nodules. 


EXPERIMENTS  103 

August  8.  Killed.  Right  testis  shows  a  whitish  irregular  area  2  or  3  mm.  in 
diameter,  and  not  characteristic  of  the  verruga  lesion.  Left  testis  shows  a  similar 
opaque  whitish  area  which  is  also  not  characteristic.     Testes  in  Zenker's  solution. 

Rabbit  No.  55.  —  Testes  inoculation,  2d  transfer  (syringe). 

July  22,  1913.  Both  testes  inoculated  with  0.5  c.c.  of  a  suspension  of  portion  of 
lesion  in  left  testis  of  Rabbit  No.  10. 

July  26.     11.11  A.M.    Testes  small,  normal. 

Aug.  3.  Definite  nodule  in  right.    Slightly  smaller  nodule  in  left 

testis. 

August  8.  Killed.  Left  testis  shows  an  elongated  well-defined  nodule  of  firm 
consistence,  pale  yellow  color,  measures  5X2  cm.  and  extends  transversely  in  the 
testis.  Right  testis  presents  a  readily  palpable  nodule.  Other  organs  appear 
normal.  Tissues  in  Zenker's  solution.  In  the  middle  of  the  testis  on  section  there 
is  a  yello^vish  nodular  mass  measuring  3.5  mm.  in  diameter.  It  is  well  marked  off 
from  the  surrounding  testicular  tissue  and  protrudes  for  a  distance  of  1  mm.  above 
the  surrounding  tissue.  It  is  firm  to  the  touch  and  the  surface  is  moist.  A  sus- 
pension of  a  portion  of  the  lesion  inoculated  into  Rabbit  No.  70.  Fresh  and 
stained  microscopical  preparations  reveal  no  microorganisms.  Cultures  from  the 
lesion  remain  sterile. 

Rabbit  No.  66.  —  Testes  inoculation,  2d  transfer  (syringe). 

July  27,  1913.  Both  testes  inoculated  with  0.5  c.c.  of  suspension  from  testis 
lesion  of  Rabbit  No.  11. 

August  3.     No  nodules. 

August  8.  Killed.  Left  testis  shows  small  yellowish  discoloration.  No 
characteristic  lesion.  Right  testis  shows  a  small  yellowish  area.  Other  organs 
appear  normal.     Tissues  in  Zenker's  solution. 

Rabbit  No.  57.  —  Testes  inoculation,  3d  transfer  (syringe). 

July  29,  1913.  Both  testes  inoculated  with  0.5  c.c.  suspension  of  lesion  from 
left  testis  of  Rabbit  No.  46  ground  in  salt  solution.  This  rabbit  shows  marked 
oedema  of  the  prepuce. 

August    3.     Testes  normal. 

August  28.  Killed.  Organs  appear  normal.  Both  testes  show  a  small  whitish 
firm  nodule,  the  largest  measuring  about  2  mm.,  the  other  1.5  mm.  These  are 
ground  in  salt  solution  and  a  portion  of  the  resulting  suspension  inoculated  into 
four  rabbits,  Nos.  78  to  81.  One  monkey,  No.  56,  also  inoculated  with  portion  of 
same  suspension.     No  tissue  saved. 

Rabbit  No.  58.  —  Testes  inoculation,  3d  transfer  (syringe). 

July  29,  1913.  Both  testes  inoculated  with  0.5  c.c.  suspension  of  lesion  of  left 
testis  of  Rabbit  No.  46  ground  in  salt  solution. 

August  3.  Testes  normal. 

Sept.  1.  Found  dead,  considerably  decomposed.  Both  testes  placed  in 
Zenker's  solution. 

Rabbit  No.  59.  —  Testes  inoculation,  3d  transfer  (syringe). 

July  29,  1913.     Both  testes  inoculated  with  0.5  c.c.  suspension  of  lesion  of  left 
testis  of  Rabbit  No.  46,  ground  in  salt  solution. 
Augu.st  3.    No  nodules. 

Sept.  1.  Found  dead,  considerably  decomposed.  Both  testes  placed  in  Zen- 
ker's solution. 


104  VERRUGA  PERUVIANA 

Rabbit  No.  60.  —  Testes  inoculation,  3d  transfer  (syringe). 

July  29,  1913.  Both  testes  inoculated  with  0.5  c.c.  suspension  of  lesion  of  left 
testis  of  Rabbit  No.  46,  ground  in  salt  solution. 

August  3.    No  nodules. 

Sept.    16.    Rabbit  thin  and  scrawny.    Testes  small,  negative. 

Sept.  30.  Killed.  Both  testes  small,  neither  shows  any  lesion,  animal  rather 
thin.     All  organs  appear  normal.     No  tissue  saved. 

Rabbit  No.  61 .  —  Culture  inoculation  into  testes. 

July  30,  1913.  Inoculated  right  testis  with  0.5  c.c.  of  cultures  Nos.  27,  28. 
Only  right  testis  found. 

August  3.     Testes  small,  normal. 

August  28,  1913.  Killed.  Testes  small,  right  somewhat  injected.  Neither 
shows  any  definite  lesion.  Lungs  show  numerous  grayish  spots  varying  from  mere 
points  to  rounded  areas  0.5  imn.  in  diameter,  some  are  confluent  and  form  larger 
areas  of  2  or  more  mm.  Certain  of  these  elevate  the  surface  of  the  pleura  slightly. 
The  liver  shows  irregular  grayish  areas  with  scar  tissue  near  the  ventral  border  and 
two  cysticercus  cysts  found  in  the  omentum.     Tissue  in  Zenker's  solution. 

Rabbit  No.  62.  —  Culture  inoculated  into  testes. 

July  30,  1913.  Inoculated  both  testes  with  0.5  c.c.  of  cultures  Nos.  27  and  28 
(q.v.). 

August  3.     Testes  normal. 

August  28.  Killed.  Both  testes  appear  normal.  Liver,  spleen,  lungs,  and 
kidneys  are  normal.     No  tissue  saved. 

Rabbit  No.  69.  —  Testes  inoculation,  3d  transfer. 

August  8,  1913.  Both  testes  inoculated  with  lesion  from  Rabbit  No.  55,  ground 
in  salt  solution. 

September  16.    Right  testis  small,  no  nodule.    Left  testis  not  found. 

September  30.  Killed.  Testes  both  very  small  and  soft;  neither  shows  any 
lesions.  White  spots  of  0.5  to  1  mm.  or  more  on  appendix.  Other  organs  appear 
normal.    No  tissue  saved. 

Rabbit  No.  70.  —  Testes  inoculation,  3d  transfer. 

August  8,  1913.  Both  testes  inoculated  with  two  of  the  reddish  nodules  from 
omentum  of  Dog  No.  2. 

August  27.  Dead.  Both  testes  appear  nonnal.  Whitish  exudate  of  soft 
consistency  in  the  pericardium  around  the  large  vessels  and  the  mediastinal  tissue, 
oedematous  and  forming  a  soft  clot  ventral  and  anterior  to  the  heart.  The  liver 
shows  numerous  grajdsh,  irregular  areas  on  its  surface.  Spleen  is  not  markedly 
enlarged  but  somewhat  rounded.    Rabbit  otherwise  negative.    No  tissue  saved. 

Rabbit  No.  7^.  —  Testes  inoculation,  3d  transfer. 

August  9, 1913.  Both  testes  inoculated  with  material  from  lesion  of  Rabbit  No.  31. 

August  25.  Found  dead  in  afternoon.  Shght  post  mortem  decomposition. 
Right  testis  presents  on  surface  a  minute  translucent  red  nodule.  On  section  no 
lesion  found  in  interior  of  either.  Lungs  show  reddish  areas  which  contain  air 
throughout.  Nasal  mucosa  shows  red  shiny  exudate  with  general  reddening  of 
mucosa. 

Rabbit  No.  75.  —  Testes  inoculation,  3d  transfer. 

August  9,  1913.    Both  testes  inoculated  with  material  from  Rabbit  No.  31. 

September  16.  Tip  of  right  scrotum  presents  a  thick  crust  with  grayish  pus 
underlying  it.  On  pressure  a  considerable  quantity  of  grayish  pus  is  expressed; 
testis  not  found.    Left  testis  presents  no  nodule. 


EXPERIMENTS  105 

September  30.  Killed.  Right  testis  for  the  most  part  destroj'ed,  epididymis 
shows  a  small  bright  yellow  area  of  necrosis.  Left  testis  shows  a  single  rather  dense 
lobule,  no  characteristic  lesion.  Four  lesions  of  cysticercus  in  omentum.  Few 
old  scars  in  the  liver.     Other  organs  normal.     No  tissue  saved. 

Rabbit  N^o.  76.  —  Testes  inoculation,  3d  transfer. 

August  9,  1913.     Both  testes  inoculated  with  material  from  Rabbit  No.  31. 

September  16.     Right  testis  negative.     Left  negative. 

September  18.  Found  dead.  Considerable  discoloration  of  abdominal  wall, 
the  surface  of  the  right  testis  shows  a  minute  elevation,  the  interior  appears 
normal  throughout.     Left  testis  appears  normal.     No  tissue  saved. 

Rabbit  No.  77.  —  Testes  inoculation,  3d  transfer. 

August  9,  1913.     Both  testes  inoculated  with  material  from  Rabbit  No.  31. 

September  16.     Both  testes  negative. 

September  30.  Killed.  Right  testis  shows  a  rather  diffuse  whitish  coloration, 
but  no  characteristic  lesion.  The  left  testis  presents  a  readily  palpable  nodule  in 
the  interior.  The  liver  presents  two  nodules,  one  measuring  9  cm.,  the  other  1.3 
cm.,  of  spherical  form  and  firm  consistence.  On  incision  they  are  found  to  consist 
of  tough  sacs  containing  firm,  dry  caseous  material.  No  acid-fast  baciUi  found  in 
the  stained  smear.  Lungs  normal.  Spleen  of  small  size,  shows  no  nodules.  Other 
organs  appear  normal.  Tissue  in  Zenker's  solution.  Left  testis  lesion  ground  in 
salt  solution  and  used  for  injection  of  two  rabbits,  Nos.  98  and  99.  (Table  i, 
page  122.)  Smears  from  nodule  in  liver  stained  by  Loeffler's  methylene  blue  show 
no  bacteria. 

Rabbit  No.  73.  —  Testes  inoculation. 

August  9,  1913.  Both  testes  inoculated  with  a  suspension  of  lesion  from  human 
lesion  produced  by  experimental  inoculation.     Right  0.5  c.c,  left  a  few  drops  only. 

September  3.  Found  dead.  Right  testis  presents  a  small  yellowish  area 
several  mm.  across.  No  lesion  seen  in  left  testis.  Other  organs  normal.  Tissue 
in  Zenker's  solution.  Microscopical  preparation  and  culture  from  the  lesion  in 
right  testis  show  no  bacteria. 

Experiments  with  Case  16 

Clinical  Features.  —  Patient  seen  in  Lima,  at  Dos  de  Mayo  Hospital, 
male,  aged  fifteen  years.  Has  worked  in  the  verruga  zone  for  several 
months  during  the  present  year,  but  has  not  lived  with  any  case  of  verruga. 
Had  malaria  one  and  one-half  years  ago,  and  again  six  months  ago.  No 
history  of  fever  at  onset  of  present  illness.  Physical  examination  of  the 
chest  reveals  nothing  abnormal.  The  spleen  is  not  palpable,  and  the  liver 
not  enlarged.  The  inguinal  and  epitrochlear  glands  are  palpable.  Physi- 
cal examination  is  otherwise  negative.  There  are  no  parasites  observed  in 
the  blood.  The  red  blood-corpuscles  number  3,000,000;  the  white  blood- 
corpuscles  4,200;  hemoglobin  65  per  cent  (Sahli).  The  Wassermann  re- 
action is  positive.  The  urine  is  normal.  There  are  a  number  of  lesions  upon 
the  skin.  Over  the  right  elbow  there  is  a  cherry-like  nodule  measuring 
about  2.5  cm.  in  diameter  and  1.5  cm.  in  height.  A  portion  of  this  was 
previously  excised  for  a  vaccination  and  inoculation  experiment  performed 


106  VERRUGA  PERUVIANA 

on  July  9.  In  the  left  iliac  region  another  cherry-red  papule  measuring 
6  mm.  in  diameter  is  present.  In  addition  there  are  three  miliary  lesions 
on  the  right  forearm  consisting  of  papules  measuring  from  1  to  2  mm.  in 
diameter,  and  two  on  the  left  leg.  There  are  also  two  subcutaneous  nodules 
measuring  from  5  to  8  mm.  in  diameter  beneath  the  skin  of  the  right  fore- 
arm, and  several  over  the  tibia  of  each  leg. 

On  August  3  the  small  papule  situated  on  the  abdomen  in  the  left  iliac 
region,  measuring  about  4  mm.  in  diameter,  was  removed  with  scissors, 
and  also  the  mulaire  lesion  situated  on  the  right  elbow,  after  disinfection 
of  the  skin.  Microscopical  preparations,  fresh  and  stained,  from  the  cut 
surface  of  the  base  of  the  mulaire  lesion  and  of  the  papule  did  not  reveal 
any  microorganisms.  Cultures  from  the  interior  through  the  base  of  the 
mulaire  lesion  after  burning  of  the  surface,  remained  sterile.  A  portion 
of  the  mulaire  lesion  was  preserved  in  Zenker's  solution  and  about  one- 
third  of  it  placed  in  Kaiserling's  solution.  Another  portion  was  used  in 
performing  antigen  experiments.  The  following  animals  were  inoculated 
with  portions  of  the  interior  of  the  papule  or  mulaire  lesion,  or  with  sus- 
pensions of  the  same  in  saline  solution. 

Inoculation  of  Animals 

Monkey  No.  3.  August  3,  1913.  Inoculated  on  the  right  side  of  the  thorax 
with  one  implant  from  the  papule,  and  on  the  left  side  of  the  thorax  with  one 
implant  from  the  mulaire  lesion. 

August  23.  Over  the  left  of  the  thorax  a  nodule  the  size  of  a  split  pea  is  pal- 
pable and  distinctly  visible. 

August  28.  A  portion  of  the  lesion  on  the  right  side  of  the  thorax  was  excised 
and  used  for  the  inoculation  of  Rabbits  Nos.  86  to  89,  and  Monkeys  Nos.  8  and  9 
(q.  v.). 

On  September  30  the  note  reads:  "  Scar  on  right  breast  at  present  shows  no 
redundant  tissue." 

Monkey  No.  4- —  August  3,  1913.  Inoculated  beneath  the  posterior  surface  of 
skin  of  the  left  ear,  and  beneath  the  skin  of  left  breast  with  small  trocar  implants. 
The  left  eyebrow  and  eyehd,  and  left  side  of  nose,  and  left  side  of  abdomen  were 
scarified  and  a  portion  of  the  mulaire  lesion  rubbed  in.  Soon  after  the  inoculation 
the  extremities  of  the  monkey  began  to  twitch  and  he  died  during  the  night.  No 
post  mortem  examination  was  made  during  our  absence. 

Rabbit  No.  37.  —  Testes  inoculation  (syringe). 

July  9,  1913.  Inoculated  both  testes  with  0.5  c.c.  of  a  suspension  of  interior  of 
verruga  nodule  ground  in  salt  solution.  Injection  about  two  hours  after  removal  of 
tissue. 

July  11.     12.25  P.M.  Right  of  scrotum  swollen,  with  firm  in- 

duration at  posterior  end.     Left  of  scro- 
tum normal.     Testes  normal. 
"      12.       9.30  A.M.     Temp.  105°  F.     Scrotum  swollen.     Testis  movable. 
"      15.     11.40    "  "       104°.4.     Both  sides  of  scrotum  swollen.     Yellow- 

ish white  areas  showing  through.  Left 
side  shows  slough  of  skin  1.4  cm.  in 
diameter  with  dark  red  surface. 


EXPERIMENTS  107 

July  18.     11.03  A.M.    Temp.  103°.9F.  Both  sides  of  scrotum  swollen.      Left, 

partially  gangrenous  with  laTge  dark 
crusted  area. 

July  19.  Found  dead.  Nostrils  clean.  The  right  side  of  scrotum  bluish  purple, 
swollen,  measures  3.4  cm.  in  length  and  1.5  across.  The  left  presents  a  sloughing 
area,  measuring  2.7  in  length  by  1.5.  There  is  a  considerable  loss  of  substance. 
The  border  of  the  opening  shows  whitish  cheesy  material.  The  surface  of  the  liver 
shows  a  gelatinous,  soft  film  of  exudate,  grayish  color.  There  are  irregular  opaque 
areas  showing  through  capsule  on  ventral  surface,  most  marked  near  posterior 
border.  Small  amount  of  exudate  over  surface  of  intestine  and  spleen.  (Tissue 
in  Zenker's  solution.)  Cultures  on  agar  from  the  heart,  peritoneum  and  testes. 
Smear  from  the  exudate  on  the  surface  of  the  liver.  Smear  from  the  liver  shows 
large  numbers  of  bacilli;  a  few  long  rods  and  many  very  short  ones,  almost  like 
cocci,  are  present. 

The  right  testis  on  incision  showed  the  testicular  tissue  dark  grayish,  mingled 
with  red.  Also  masses  of  tliick  creamy  whitish  material  of  puriform  appearance. 
Small  abscesses  in  the  epididymis  contain  thinner  whitish  exudate  of  milky  consist- 
ence. One  small  abscess  in  the  cord  contains  white,  creamy  material.  The  left 
testis  on  section  is  found  to  be  gangrenous  throughout,  and  is  surrounded  by 
exudate  forming  a  layer  1  mm.  or  more  in  tliickness.  Cultures  and  smears  made 
from  right  testis. 

Smear  from  the  left  testis  shows  few  bacteria,  short  baciUi  and  cocci.  Cul- 
tures made  from  left  testis  and  from  the  heart  and  from  the  peritoneum  on  agar 
develop  a  few  colonies  of  both  cocci  and  colon  bacilh  which  are  not  believed  to  bear 
any  etiological  relationship  to  the  original  verruga  lesion.  The  mulaire  tissue 
employed  for  inoculation  had  evidently  become  secondarily  infected  through  its 
surface  with  bacteria. 

Rabbit  No.  38.  —  Testes  inoculation  (syringe). 

July  9,  1913.  Inoculated  both  testes  with  0.5  c.c.  of  a  suspension  of  the  interior 
of  a  verruga  nodule  ground  in  salt  solution.  Injection  about  4|  hours  after  re- 
moval of  tissue. 

July  11.  12.25  P.M.  Rabbit's  eyes  closed.  Discharge  from  nose.  Difficulty 
in  breathing.     Testes  both  normal. 

July  11.  5.00  P.M.  Dead;  marked  rigor  mortis.  Austopsy  at  once,  tissues 
fresh.  Thin  yellowish  fluid  runs  from  nose.  Lungs,  patchy  reddening  throughout. 
Peritoneum  smooth.  Smear  nasal  discharge  shows  a  small  bacillus  and  larger 
diplococcus.  Smear  from  lung  reveals  short  bacilli  in  leukocytes.  Testes  each 
show  a  sUghtly  paler  ill-defined  area.     A  shce  fixed  in  Zenker's  solution. 

Experiments  with  Case  7 

Clinical  Features.  —  July  4,  1913.  — ■  A  child  one  and  one-half  years  of 
age.  Lives  in  San  Bartolome,  where  the  patient  was  seen.  Mother  says 
the  baby  has  had  a  very  high  fever,  which  commenced  four  months  ago. 
The  eruption  appeared  one  and  one-half  months  ago.  The  child  is  of 
sickly  appearance.  It  has  no  fever  today.  There  is  an  extensive  erup- 
tion over  the  back  of  the  hands,  forearms  and  arms,  legs  and  thighs. 
Mother  refuses  to  remove  clothes.  States  there  are  no  lesions  on  the 
trunk  and  a  complete  examination  cannot  be  made.  The  lesions  consist 
of  numerous  discrete  and  a  few  confluent  papules.     On  the  legs  they  are 


108  VERRUGA  PERUVIANA 

very  numerous  and  sometimes  closely  placed.  They  are  usually  of  the  color 
of  cherries,  or  a  little  more  pink.  In  a  few,  hemorrhages  have  occurred, 
and  these  are  of  a  dark  blue  or  black  color.  The  cherry-like  tumors  have 
a  translucent  appearance.  They  measure  from  about  1.5  mm.  to  4  mm.  in 
diameter,  and  have  a  height  from  2  to  3  mm.  There  are  a  fair  number  of 
papules  which  have  a  pink  or  gray  color,  which  do  not  measure  more  than 
0.5  to  1  mm.  in  diameter.  The  eruption  is  very  extensive  and  appears 
acute. 

Note:  —  A  blood  examination  shows  no  parasites  of  malaria  or  of  Oroya  fever. 
The  child  was  nursing  and  the  mother  would  not  permit  a  physical  examination  to 
be  made.  An  area  of  the  skin  upon  which  a  number  of  the  papules  were  situated 
was  cleansed  with  alcohol  and  ether  and  lightly  brushed  with  iodin  solution.  Four 
of  the  bright  cherry-red  papules  were  then  removed  with  sterile  forceps  and  scissors. 
These  tmnors  were  then  sheUed  from  the  skin  with  a  scalpel;  three  were  placed  in  a 
mortar  and  finely  ground  in  saUne  solution  (0.85  per  cent).  The  following  animals 
were  immediately  inoculated  with  the  suspension  thus  obtained  or  Avith  bits  of  the 
fourth  tumor. 

Inoculation  of  Animals 
Rabbit  No.  21^.  —  Testes  inoculation  by  trocar. 

July  6,  1913.  Both  testes  inoculated  by  trocar  with  bits  of  verruga  immedi- 
ately after  removal  from  child  at  San  Bartolome  (Case  7). 

July    9.     11.25  A.M.    Temp.  102°  F.      Distinct  nodule  in  central  portion  of 

right  testis.     Also  in  central  portion  of 
left. 
"      11.     11.50    "  The  puncture  point  shows  small  crust. 

Surface  of  right  testis  shows  a  dark 
bluish  discoloration  2  to  3  mm. ;  definite 
firm  nodule  in  central  ventral  portion. 
Left  shows  minute  nodule  near  crust  of 
skin.  Also  small  firm  nodule  in  central 
portion  of  testis. 
"      15.     11.12    "  Both  show  small  yellow  whitish  nodule 

beneath    skin    at    point    of    puncture. 

Both  show  small  nodule  in  the  interior. 

"      18.     11.50    "  Both  nodules  beneath  skin  no  larger. 

Both  show  nodules  palpable  in  interior. 
"      21.     11.22    "  Definite  small  nodules.     No  increase  in 

either  of  these  or  in  the  nodules  in  the 
skin. 
"     26.     12.17  p.m.  Right,  small  nodule  within.     Left,  large 

definite  nodule  plainly  felt.     Subcutane- 
ous nodule  in  scrotum  not  increasing. 
"     30.  Right,  small  nodule.     Left,  larger  defi- 

nite palpable  nodule. 
Aug.  3.  Right,  small  nodule.     Left,  larger  defi- 

nite palpable  nodule. 
August  3.   Killed.    Left  testis  presents  lesions  measuring  2  to  3  mm.  in  diameter. 
Ground  in  mortar,  and  inoculated  into  Rabbit  No.  65.     Organs  appear  normal. 
Small  nodules  in  right  testis  placed  in  Zenker's  solution. 


EXPERIMENTS  109 

Microscopical  preparations  from  the  nodules  in  the  testis  show  no  spirochaetae 
or  other  visible  microorganisms.     Cultures  remained  sterile. 

Rabbit  N^o.  25.  —  Testes  inoculation  by  trocar. 

July  6,  1913.  Both  testes  inoculated  by  trocar  with  bits  of  verruga  imme- 
diately after  removal  from  child  at  San  Bartolome.  This  rabbit's  testes  were  very 
small  for  this  mode  of  inoculation. 

July    9.     11.25  A.M.  Temp.  102°.5F.    Testes  small  and  apparently  normal. 
"      11.     11.55    "  Testes  very  small;    minute  nodule  in 

left. 
"      15.     11.10    "  No  definite  nodule.    Testes  small,  unsat- 

isfactory examination. 
"      18.     11.52    «  No  definite  nodule. 

"21.  "  Small  hard  nodule  at  posterior  tip  of  left 

testis. 
"      26.     12.20  P.M.  Small  hard  nodule  at  posterior  tip  of  left 

testis. 
"     30.  SmaU.    Negative. 

Aug.  3.  «  « 

August  8.  Killed.  Left  testis  shows  a  nodule  2  to  3  mm.  in  diameter,  grayish 
with  dark  marking,  near  periphery.  Right  epididymis  shows  a  similar  nodule, 
dark  gray,  measuring  about  2  mm.  in  diameter.  Liver  shows  two  coccidial  nodules. 
Other  organs  appear  normal.  Tissues  in  Zenker's  solution.  No  culture  taken. 
Microscopical  preparations  show  no  spirochaetae  or  other  visible  microorganisms. 

Rabbit  No.  26.  —  Testes  inoculation  by  syringe. 

July  6,  1913.     Both  testes  inoculated  with  0.5  c.c.  of  suspension  of  verrugas 

ground  in  salt  solution  as  soon  as  possible  after  removal  from  child  at  San  Bartolome. 

July    9.     11.30  a.m.    Temp.  102°  F.     Left     testis    homogeneous,     right    not 

found. 
Right  testis  not  found. 

"  "      does  not  slip  down.     Left 

normal. 

Right  testis  does  not  slip  down.      Left 
normal. 

Neither  testis  shows  any  nodule. 
Neither  testis  shows  any  nodule.    Right, 
small,  flabby. 
"     30.  Left    deformed.     Neither    shows    any 

nodule.     Right,  small,  flabby. 
Aug.    3.  Bitten  on  back.      Neither  shows  any 

nodule.  Right,  small,  flabby. 
August  8.  Killed.  Left  testis  appears  normal  throughout.  Right  testis  atro- 
phied, presents  at  both  anterior  and  posterior  poles  a  yellowish  opaque  area.  Near 
the  latter  is  a  small  translucent  bulging  area.  Rabbit's  back  has  been  bitten,  and 
there  is  a  large  abscess,  with  considerable  hemorrhage  beneath  skin.  Spleen  small. 
Tissues  in  Zenker's  solution.     No  cultures. 

Rabbit  No.  27.  —  Testes  inoculation  by  syringe. 
July  6,  1913.      Both  testes  inoculated  with  0.5  c.c.  of  suspension  of  verrugas 
ground  in  salt  solution  as  soon  as  possible  after  removal  from  child  at  San  Bartolom^. 
(Verruga  Ca.se  7.) 

July    9.     11.30  a.m.  Temp.  101°  .8  F.  Both  testes  homogeneous  except  slight 

induration  anterior  end  of  left. 


11. 

12.00  M. 

15. 

11.15  A.M. 

18. 

11.54     " 

21. 

26. 

12.23  P.M. 

July  11. 

11.55  A.M. 

«      15. 

11.17    " 

"      18. 

11.55    " 

"     21. 

11.30    " 

«     26. 

12.22  P.M. 

110  VERRUGA  PERUVIANA 

Testes  both  normal. 

but  firm  and  tense. 

u  u  u  u 

Right  testis  swollen  posteriorly.     Left 
negative. 
"     30.  Right  testis  swollen  posteriorly.     Left 

apparently  negative. 
August  8.      Killed.      Both  testes  small.      Shghtly  soft  throughout.      Slight 
excess  of  fluid  in  peritoneum.     Organs  appear  normal.     No  cultures.     No  tissue 
saved. 

Rabbit  No.  65.  —  Testes  inoculation,  2d  transfer. 

August  3,  1913.  Inoculated  both  testes  with  suspension  of  material  from 
Rabbit  No.  24. 

September  16.  Right  testis  small  and  soft,  negative.  Left,  small  and  soft, 
negative.     Rabbit  thin  and  fur  dirty. 

September  25.  Found  dead.  Considerable  post  mortem  decomposition. 
Right  testis  normal  throughout;  left  also  normal.  A  minute  focus  of  pneumonia 
in  lung.     No  tissue  saved. 

EXPEKIMENTS    WITH    CaSE    17 

On  July  10  Case  17,  which  showed  an  abundant  verruga  eruption  (page 
73  for  description  of  the  lesions  present)  was  visited.  After  disinfection 
of  the  skin  with  soap  and  water,  alcohol,  ether,  and  iodin,  a  number  of  the 
lesions  were  removed.  One  of  the  subcutaneous  nodules  situated  just 
superiorly  and  exteriorly  to  the  right  patella  was  first  excised.  Micro- 
scopical preparations  were  made  from  the  interior  of  this  nodule  and  also 
a  number  of  cultures  upon  various  media.      (Page  111.) 

Two  guinea  pigs  (Nos.  1  and  2,  page  113),  and  two  rabbits  (Nos.  42 
and  43,  page  112)  were  inoculated  with  a  suspension  of  a  portion  of  the 
subcutaneous  nodule  ground  up  in  saline  solution.  Rabbits  Nos.  42  and 
43  were  inoculated  in  both  testes.  Guinea  Pig  No.  1  was  inoculated  intra- 
peritoneally,  and  Guinea  Pig  No.  2  intraperitoneally  and  into  the  right 
testis. 

Two  small  papules,  measuring  4  and  5  mm.  in  diameter,  and  2  or  3  mm. 
in  height,  situated  below  the  right  knee,  were  also  excised  and  hardened 
for  section.  A  portion  of  the  subcutaneous  nodule  was  also  hardened  for 
section. 

A  third  tumor,  of  a  cherry-red  color,  measuring  12  mm.  in  diameter, 
and  8  mm.  in  height,  was  also  excised  from  the  toe.  Much  sticky  serum 
exuded  from  the  cut  surface,  which  was  very  soft.  Microscopical  prepara- 
tions, cultures  and  inoculations  were  also  made  from  this  nodule  (page 
111),  as  well  as  a  portion  hardened  and  saved  for  histological  study. 

Rabbits  Nos.  39  and  40  (page  111)  were  inoculated  into  both  testes,  and 
No.  41  intraperitoneally,  with  a  portion  of  the  suspension  of  this  nodule 
in  saline  solution. 


EXPERIMENTS  111 

Before  the  removal  of  the  tumors  the  skin  was  cleansed  with  alcohol, 
ether  and  iodin.  Disinfection  over  the  nodule  from  the  toe  was  not  as 
thoroughly  performed. 

Microscopical  aiid  cultural  results.  —  The  study  of  the  microscopical 
preparations  from  the  excised  papule  and  subcutaneous  nodule  in  both 
the  fresh  and  stained  condition  did  not  reveal  any  spirochaetae  or  other 
microorganisms.  Wright's,  Giemsa's,  and  methylene  blue  stain  were 
emploj-ed.  In  the  microscopical  preparation  made  from  the  lesion  re- 
moved from  the  toe,  a  fair  number  of  short  bacilli  were  present.  Cultures 
were  made  from  the  different  lesions  and  the  following  results  obtained. 

Two  cultures  were  made  upon  alkaline  agar,  one  from  the  papule  and 
one  from  the  subcutaneous  nodule.  Both  of  these  remained  sterile  after 
two  weeks.  Four  cultures  were  made  from  the  interior  of  the  papule  in 
ascitic  fluid  covered  with  paraffin  oil.  After  two  weeks  the  fluid  was  still 
clear  and  microscopical  examination  showed  no  microorganisms.  Sub- 
cultures on  agar  gave  no  growth.  One-half  cubic  centimeter  of  each  of 
two  of  these  cultures  was  inoculated  into  the  testes  of  Rabbit  No.  63 
(q.  v.).  No  lesion  resulted.  Four  cultures  were  made  from  the  subcuta- 
neous nodule  in  ascitic  fluid  agar  +  rabbit's  testis  +  a  fragment  of  the 
subcutaneous  nodule  in  the  center  and  covered  with  paraffin  oil.  Three  of 
these  cultures  remained  clear  and  showed  no  cloudiness  after  two  weeks. 
The  fourth  culture  showed  marked  cloudiness  after  about  a  week,  and 
microscopical  examination  revealed  in  the  culture  a  large  bacillus,  appar- 
ently a  contamination  from  the  air.  Two  cultures  from  the  subcutaneous 
nodule  in  ascitic  agar  +  rabbit's  kidney  +  a  fragment  of  the  subcutaneous 
nodule  in  the  center  (no  paraffin  oil)  were  made.  One  culture  remained 
clear  after  two  weeks,  and  microscopical  examination  showed  no  visible 
microorganism;  the  other  became  cloudy  shortly  after  the  inoculation, 
and  microscopical  examination  revealed  a  large  pleomorphic  bacillus.  One 
culture  was  made  from  the  papule  removed  from  the  toe  in  ascitic  fluid 
agar  -f-  rabbit's  testis  +  a  fragment  of  lesion  from  the  toe.  The  media 
became  cloudy  and  showed  a  growth  with  production  of  gas  after  nine 
days.  This  organism  present  was  a  large  bacillus  and  gave  a  heavy  white 
growth  on  agar.  One  other  culture  made  from  the  lesion  of  the  toe  in 
ascitic  fluid  +  rabbit's  testis  +  a  fragment  of  the  lesion  of  the  toe  also 
became  cloudy  and  showed  the  same  bacillus  after  about  a  week.  The 
bacteria  found  in  the  three  cultures  in  question  obviously  had  nothing  to 
do  with  the  primary  etiology  of  verruga. 

Inoculation  of  Animals 

Rabbit  No.  39.  — Testes  inoculation  (syringe). 

July  10,  1913.  Inoculated  both  testes  with  0.5  c.c.  of  a  suspension  of  interior 
of  lesion  removed  from  foot  of  Case  17. 

July  15.     11.45  a.m.    Testes  large,  flabby,  normal. 


112  VERRUGA  PERUVIANA 

July  18.     12.04  P.M.     Right  side  of  scrotum  swollen,  tense.      Left  side  less 
swollen.     Testis  somewhat  tense. 
"     21.     11.54  A.M.     Right  side  of  scrotum  greatly  swoUen.     Left  side  slightly 

swoUen.     Testis  negative. 
"      26.     12.39  P.M.     Right  side  of  scrotum  tense.     Left  side  consists  of  firm 

mass. 

"     29.  Right  side  of  scrotum  —  hard  mass.     Left  side  shps,  but 

nodular  throughout.     Animal  sickly,  cold  and  does  not 

eat. 

July  30.     Killed.     Cecum  shows  numerous  1  mm.  white  lesions  beneath  serous 

membrane  of  appendix  and  ileocecal  pouch.     Kidneys  show  a  few  red  spots  1  mm. 

or  less.     Liver,  several  whitish  rounded  nodules  2  to  3  mm.  (cocci dial  infection). 

Testis,  right,  large  with  whitish,  opaque  masses  and  dry  caseous  exudate  on  surface. 

On  section  large  masses  of  soft,  dry,  caseous  material.     Epididjrmis  shows  multiple 

regularly  distributed  areas  from  which  thick  pus  exudes.     Left  testis  shows  multiple 

mihary  abscesses.      Tissues  in  Zenker's  solution.      Stained  smear  from  appendix 

lesion  shows  a  variety  of  bacteria  in  and  between  cells.     Smears  from  the  lesions 

of  the  left  testis  show  a  bacillus.     This  organism  is  not  acid-fast  and  decolorizes 

by  Gabbett's  solution.      A  culture  from  the  lesion  in  the  testis  shows  the  same 

organism.      In  glucose  agar  it  produces  gas  and  appears  to  belong  to  the  colon 

group. 

Rabbit  No.  Jfi-  —  Intravenous  inoculation. 

July  10,  1913.  Inoculated  in  ear  vein  with  1  c.c.  of  suspension  of  same  material 
as  used  in  Rabbit  No.  39. 

July  15.  11.45  a.m.  Temp.  105°. 3  F.  Blood  shows  no  parasites  (Giemsa  stain). 
"      18.     12.07  p.m.  "       lOr.l. 

July  21.  Rabbit  found  dead.  No  rigor  mortis  present.  Body  cold.  Peri- 
toneal cavity  contains  approximately  50  c.c.  of  clear  watery  fluid,  with  soft  gela- 
tinous clot.  Spleen  small.  Kidneys  injected.  Two  or  three  small  grayish  spots 
in  the  lung  about  1  mm.  across.  Intestinal  infection  with  coccidia.  Smear  from 
peritoneal  fluid  stained  with  Loeffler's  methylene  shows  no  bacteria.  Culture  on 
agar  from  the  heart  is  negative.     No  culture  taken  from  spleen. 

Rabbit  No.  41-  — Intraperitoneal  inoculation. 

July  10,  1913.  Injected  1.25  c.c.  of  same  material  used  in  Rabbits  Nos.  39  and 
40  into  the  peritoneal  cavity. 

July  15.     12  M.     Temp.  102°.8F. 

"      18.     12.08  p.m.  "       103°.7. 

"     21.     11.52  a.m.  "       102°. 

August  8.  Killed.  Peritoneum  and  aU  organs  appear  normal.  No  tissue 
saved. 

Rabbit  No.  1^.2.  —  Testes  inoculation  (syringe). 

July  10,  1913.     Inoculated  both  testes  with  0.5  c.c.  of  a  suspension  of  subcu- 
taneous nodule  from  knee  Case  17,  ground  in  salt  solution. 
July  15.     12.02  P.M.     Testes  small,  normal. 

"      18.     12.10    "       Temp.  101°.5F.     Testes  small,  normal. 

"     21.     11.50  a.m.  "       101°.6. 

"     26.     12.40  p.m. 

"     29.  "  "  " 

Aug.    3.  "  «  " 

August  8.  Killed.  A  small  nodule  (yellowish)  near  surface  of  left  testis.  Right 
negative.     Left  testis  placed  in  Zenker's  solution. 


EXPERIMENTS  113 

Rabbit  N'o.  43.  —  Testes  inoculation  (sjTinge). 

July  10.  Inoculated  both  testes  with  0.5  c.c,  of  a  suspension  of  nodule  from 
knee  of  Case  17. 

July  15.     12.03  P.M.     Testes  firm,  normal. 
"'    18.     12.12    "       Temp.  102°.6F.     Testes  homogeneous. 

July  19.  Found  dead.  Body  still  warm,  slight  rigor.  On  opening  abdominal 
cavity  about  two  dozen  cysticercus  cysts,  measuring  from  about  4  to  8  mm.  in 
diameter,  distributed  over  the  omentum.  The  liver  shows  glistening,  whitish 
nodules  distributed  near  the  ventral  border  of  the  left  central  lobe.  On  section 
some  of  the  lesions  are  rather  dry  and  cheesy.  The  lungs  are  of  a  bright  red  color, 
showing  no  lesions  or  discolorations.  The  fur  is  moist  about  the  nostrils  and 
mouth.  Nasal  mucosa  clean.  Tissue  in  Zenker's  solution.  Cultures  from  the 
heart  show  after  fortj^-eight  hours  one  large,  white  colony,  evidently  a  contamina- 
tion from  the  air.  On  longitudinal  incision  both  testes  appeared  normal  through- 
out. 

Guinea  Pig  No.  1.  —  Intraperitoneal  inoculation  (syringe). 

July  10,  1913.     Inoculated  1  c.c.  of  suspension  of  nodule  from  knee  of  Case  17. 

July  15.     12.04  P.M.     Appears  normal. 

"      18.     12.18    " 

"     21.  Negative. 

"     26. 

Guinea  Pig  No.  2.  —  Intraperitoneum  and  testes  inoculation. 

July  10,  1913.  Inoculated  intraperitoneally  with  1  c.c.  of  suspension  of  knee 
nodule  from  Case  17.  The  right  testis  inoculated  with  0.5  c.c.  of  suspension  of 
same  material. 

July  15,     12.04  P.M.     Appears  normal.     No  nodules  in  testes. 
"'    18.     12.18    "  "  "  "         "         "       « 

"     21.     11.57  A.M.    Testis  inflammation,   uneven  induration.    Animal  not 
fat. 

July  23.  10.00  .\.M.  Animal  died  during  night.  Peritoneum  smooth,  but  dry 
and  sticky.  Omentum  yellowish  and  opaque.  All  of  left  lung  consolidated  except 
apex  —  dark  red.  Smears  from  peritoneum,  omentum,  spleen  and  heart's  blood. 
Those  from  the  peritoneum  and  heart's  blood  show  numerous  bacilh.  They  are 
not  acid-fast  and  decolorize  easily  with  Gram's  stain.  Cultures  from  lower  part 
of  peritoneal  cavity,  omentum,  and  heart's  blood  on  agar. 

July  30.  The  culture  from  the  peritoneal  cavity  and  omentum  shows  numerous 
large,  white  colonies.     This  organism  produces  gas  in  glucose  agar. 

Peritoneal  cavity  washed  out  with  5  c.c.  salt  solution,  and  injected  into  a  second 
guinea  pig  intraperitoneally. 

Further  Experiments.  —  On  July  29  there  remained  on  the  anterior  sur- 
face of  the  right  foot  a  "  tumor,  which  was  pinkish  in  color,  and  which 
measured  about  5  mm.  in  diameter.  It  was  raised  for  about  2  mm.  above 
the  surface  of  the  skin.  This  was  removed.  On  section  it  was  quite 
moist  and  not  dried.  Another  nodule,  on  the  dorsum  of  the  foot  over 
which  the  skin  had  become  dried,  and  resembled  an  onion  skin,  measuring 
about  7  mm.  in  diameter  and  5  mm.  in  height  was  also  removed.  It  was 
almost  the  color  of  the  normal  skin.  On  cut  section  it  likewise  proved  to 
be  moist  in  the  interior. 


114  VERRUGA  PERUVIANA 

"  The  eyebrows,  cheeks,  nose,  chin  and  abdomen  of  Monkey  No.  2  were 
scarified,  and  the  two  tumors  removed  thoroughly  rubbed  into  the  scari- 
fications within  fifteen  minutes  after  removal." 

Monkey  No.  2.  —  Cutaneous  inoculation. 

July  29,  1913.  Visited  Dos  de  Mayo  Hospital,  and  excised  pendulous  papule 
and  larger  soft  lesion  from  dorsum  of  right  foot  of  patient.  On  section,  interior  of 
both  were  translucent,  but  not  soft.  Monkey  scarified  on  both  brows,  sides  of  nose, 
inner  surface  of  lower  lip,  and  implants  made  of  material  of  smaller  lesion  beneath 
the  skin  of  the  posterior  surface  of  the  ears.  Multiple  scarifications  made  on  the 
belly  and  chin  with  material  from  the  larger  lesion.  A  portion  of  the  large  lesion 
and  a  portion  of  the  papule  placed  in  Zenker's  solution. 

August    3.     Face  appears  smooth;  brows  and  nose  normal. 

August  23.     Up  to  present  date  no  lesion  has  appeared  on  the  face. 

Jan.  21,  1914.     Monkey  has  remained  well,  no  eruption  has  appeared. 

Rabbit  No.  68.  —  Culture  inoculation  into  testes. 

July  30,  1913.  Inoculated  both  testes  with  0.5  c.c.  from  culture  in  ascitic  fluid 
from  miliary  lesion  Case  17. 

August    3.     Left  slightly  uneven. 

August  28.  Killed.  Both  testes  appear  normal.  Except  for  scar  along  ventral 
edge  of  liver,  organs  appear  normal.     No  tissue  saved. 

Experiments  with  Case  19 

On  the  afternoon  of  July  17th  about  twelve  papules,  measuring  from 
3  to  about  6  mm.  in  diameter  (situated  over  the  right  arm  and  shoulder, 
and  right  and  left  leg)  and  one  mulaire  lesion  measuring  11  mm.  in  diameter 
(situated  over  the  left  thigh)  were  removed  with  scissors  after  thoroughly 
cleansing  the  skin  with  soap  and  water,  alcohol,  ether  and  iodin.  The 
tumors  were  immediately  transferred  to  sterile  Petri  dishes,  placed  in  a 
sterile  container,  and  brought  to  the  laboratory.  Ten  of  these  papules 
were  then  ground  up  in  fifteen  cubic  centimeters  of  saline  solution  in  a 
mortar,  and  twelve  cubic  centimeters  of  the  solution  filtered. 

Rabbits  Nos.  48  to  51  were  inoculated  into  both  testes,  each  with  1  c.c, 
and  Dog  No.  4  into  both  testes  each  with  1  c.c.  The  three  cubic  centi- 
meters which  remained  on  the  filter  and  the  fluid  in  the  filtrate  tube  were 
then  placed  in  a  mortar  and  the  remaining  papule  ground  up,  and  three 
or  four  cubic  centimeters  of  saline  solution  added.  Rabbits  Nos.  52  and 
53  were  inoculated  with  this  suspended  residue  into  both  testes,  each  with 
0.5  c.c,  and  Dog  No.  5  into  each  testis  with  0.5  c.c.  and  with  1  c.c.  sub- 
cutaneously. 

The  mulaire  lesion  was  now  ground  up  in  salt  solution,  and  added  to  the 
remaining  papular  extract.  A  guinea  pig  was  inoculated  with  3  c.c  of  this 
suspension  intraperitoneally. 

Cultures  were  made  from  one  of  the  papules,  small  pieces  being  placed 
at  the  bottom  of  ascitic  fluid  (5  cultures)  and  in  the  middle  of  ascitic  agar 


EXPERIMENTS  115 

(4  cultures)  and  alkaline  agar  (2  cultures).  An  equal  number  of  similar 
controls  was  prepared.  One-half  of  the  cultures  in  ascitic  fluid  and  ascitic 
agar  were  covered  with  paraffin  oil.  After  twenty-four  hours  culture  tubes 
and  controls  appeared  sterile. 

Cultures  made  from  the  interior  of  the  mulaire  lesion  on  alkaline  agar 
showed  after  twenty-four  hours  about  one  hundred  colonies. 

A  culture  from  the  filtrate  used  for  inoculating  Rabbits  Nos.  48  to  51 
and  Dog  No.  4  on  agar  remained  sterile  after  seventy-two  hours. 

(The  inoculation  of  the  animals  was  performed  in  from  two  and  one-half 
to  three  and  one-half  hours  after  removal  of  the  tumors.) 

Microscopical  preparations  were  made  from  the  interior  of  the  papules 
and  from  the  cut  surface  at  the  base  of  the  mulaire  lesion.     In  the  fresh 
and  stained  preparations  from  the  papules  no  definite  microorganism  could 
be  observed.     The  most  striking  feature  was  the  occurrence  of  small, 
deeply-staining,  blue  disks  in  some  of  the  preparations.     These,  however,' 
had  not  been  observed  in  preparations  made  from  the  lesions  of  other 
cases  of  verruga.     Possibly  they  were  fragments  of  nuclei.     The  reaction 
on  the  part  of  many  of  the  cells  to  the  virus  of  the  disease  seemed  evident  in 
some  of  these  preparations,  and  many  of  them  showed  granules  of  various 
character,  staining  either  deeply  red  or  deeply  blue  with  Giemsa's  stain. 
No  spirochaetae  were  detected.     In  the  preparations  from  the  mulaire 
lesion  numerous  cocci  in  clumps,  pairs,  or  short  chains  were  observed.     An 
occasional  bacillus  was  also  seen.     In  other  preparations  these  bacilli  were 
not  acid-fast,  and  decolorized  easily  by  Gabbett's  stain.     The  culture  from 
the  mulaire  lesion  on  agar  referred  to  above  showed  at  first  only  white 
colonies.     Later  many  of  these  became  of  a  yellow  color.     Apparently 
Staphylococcus  aureus  and  albus  were  present.      The  cultures  made  from 
the  papule  in  special  media  referred  to  above  were  observed  from  July  17 
until  July  30.     During  this  time  no  evidence  of  growth  was  appreciable 
in  any  of  the  tubes.     Microscopical  preparations  made  from  the  different 
ones,  from  time  to  time,  did  not  reveal  any  visible  microorganisms,  but 
only  an  increasing  number  of  deeply  staining  granules,  probably  formed 
by  the  continual  disintegration  of  the  tissue  implanted.     Subcultures  on 
agar  from  these  tubes  revealed  no  growth.     On  July  30  two  of  the  cultures 
made  in  ascitic  fluid,  one  which  had  been  under  paraffin  oil  and  the  other 
without,  were  inoculated  into  the  testes  of  rabbit  No.  64  (q.  v.)  0.5  c.c. 
of  each  culture  being  injected  into  each  testis.      No  definite  lesions  were 
produced  by  the  inoculation. 

Inoculation  of  Animals 

Rahlrit  No.  48.  —  Testes  inoculation  with  filtrate. 

July  17,  1913.     Both  testes  inoculated  with  1  c.c.  of  filtrate  from  skin  nodules 
from  Case  19. 

July  21.  Testes  normal. 


116  VERRUGA  PERUVIANA 

July  26.  Testes  normal.     Rather  small  and  firm. 

((       on  <'  «  <t  «  «  " 

Aug.    3. 

August  8.  Killed.  Both  testes  negative.  Lungs  show  numerous  discrete 
spots  of  pigmentation.  One  testis  shows  a  minute  translucent  spot  on  surface. 
Not  visible  on  section. 

Rabbit  No.  49-  —  Testes. inoculation  with  filtrate. 

July  17.  Both  testes  inoculated  with  1  c.c.  of  filtrate  from  skin  nodules  from 
Case  19. 

July  21.  Testes  normal. 

"     26.     12.50  P.M.         "        large,  moderately  soft,  normal. 

((       on  ('  li  "  "  « 

Aug.    3. 

Rabbit  No.  50.  —  Testes  inoculation  with  filtrate. 

July  17.  Both  testes  inoculated  with  1  c.c.  of  filtrate  from  skin  nodules  from 
Case  19. 

July  21.    Normal  testes. 
«     26.          "  " 

"     29.  "  " 

Aug.    3. 

August  8.  Killed.  Both  testes  show  a  minute  translucent  spot  on  surface 
(point  of  puncture).  Liver  an  area  of  necrosis,  and  exudate  on  surface.  Cysts 
and  necrosis  in  lobes. 

Rabbit  No.  51.  —  Testes  inoculation  with  filtrate. 

July  17.  Both  testes  inoculated  with  1  c.c.  of  filtrate  from  skin  nodules  from 
Verruga  19. 

July  21.     Testes  both  normal. 
"     26.         "  "  " 

<i        oy  «  «  « 

Aug.    3. 

August  28.  Killed.  Both  testes  appear  normal  but  of  rather  small  size. 
Liver  shows  several  whitish  masses.  Organs  otherwise  appear  normal.  No 
tissue  saved. 

Rabbit  No.  52.  —  Testes  inoculation,  unfiltered  virus. 

July  17.  Both  testes  inoculated  with  0.5  c.c.  of  the  suspension  of  skin  nodules 
from  Case  19,  portion  left  upon  filter  resuspended.  Another  skin  nodule  was 
also  ground  and  added  to  make  up  for  dilution. 

July  21,    Both  testes  normal. 
«     26.        "  "      rather  firm  as  though  overlooked  previously;  inflamed. 

Definite  firm  nodule  in  left. 
"     29.     Definite  firm  nodule  in  right.     Left,  not  possible  to  get  down. 

August  3.  Large  rounded  nodule  in  both.  Animal  very  emaciated.  Killed  by 
blow.  The  rabbit  was  weak  and  cold  and  extremely  emaciated  before  being  killed. 
No  abnormality  noted  to  account  for  this  condition.  Testes  show  perfectly  opaque 
lesion  yellowish  in  greater  portion;  slightly  grayish  pink  toward  periphery.  •  Tissue 
in  Zenker's  and  Kaiserhng's  solutions.  A  portion  suspended  and  inoculated  into 
Rabbits  Nos.  66,  67,  and  68  (q.  v.).  Microscopical  preparations  from  the  lesion 
of  the  testis  show  no  microorganisms.  Cultures  on  agar  developed  two  large, 
white  colonies  of  a  large  bacillus,  perhaps  a  contamination  from  the  razor  used  in 
sectioning  the  lesion. 


EXPERIMENTS  117 

Rabbit  No.  33.  —  Testes  inoculation  with  unfiltered  ^^rus. 

July  17.  Both  testes  inoculated  with  0.5  c.c.  suspension  of  skin  lesions  from 
Case  19  removed  from  filter.  An  additional  skin  lesion  was  added  to  make  up  for 
dilution. 

July  21.     Both  testes  normal. 
"     26.     Right  no  nodule.     Left  definite  firm  nodule. 
"     29.     Both  large,  firm  nodules.     Prepuce  ulcerated  and  swollen. 

Aug.    3.     Both  testes  show  a  large  firm  nodule. 

August  8.  The  left  testis  excised  by  operation.  On  section  it  shows  a  rounded, 
well-defined  lesion  7X6  mm.  across,  and  bulging  from  surface.  In  general  it  i.s 
of  a  pale  pink  color,  similar  to  the  color  of  the  testis,  but  mingled  with  it  are  minute 
red  pomts,  and  the  central  portion  shows  firm,  dry,  whitish,  necrotic  tissue.  Speci- 
men photographed.  A  microscopical  preparation  shows  no  bacteria.  A  culture 
on  agar  remained  sterile.  Lesion  ground  in  saline  solution  and  a  portion  of  the 
suspension  inoculated  into  Rabbits  Nos.  71  and  72  (q.  v.). 

Dog  No.  4.  —  Testes  inoculation  wdth  filtrate. 

July  17,  1913.     Both  testes  inoculated  with  1  c.c.  filtrate  of  suspension  of  skin 
lesions  from  Case  19  witliin  two  hours  after  removal. 
July  21.  Testes  hard,  nothing  abnormal. 

"     26.     12.59  p.m. 

"     29.  «  «  «  « 

Aug.    3.  "  «  «  « 

August  9.  Killed.  Testes  firm  and  present  no  macroscopic  lesions.  All  organs 
appear  normal.     No  tissue  saved. 

Dog  No.  5.  —  Testes  and  subcutaneous  inoculation. 

July  17,  1913.  Inoculated  left  testis  with  0.5  c.c.  of  suspension  of  skin  lesions 
from  case  used  in  last  two  rabbits  (Nos.  52  and  53),  1  c.c.  of  same  material  inocu- 
lated subcutaneously  over  right  thorax. 

July  21.     Skin  slight  subcutaneous  oedema.     Testes  hard. 

26.     Thorax  shows  a  tliickening  1.5  cm.  in  diameter  with  some  diffuse 

swelling  of  the  surrounding  tissue. 
29.     Subcutaneous  sweUing  more  localized.     Testes  negative. 

Aug.     3.     Subcutaneous  swelling  smaller.     Testes  nothing  definite. 

August  9.  Killed.  There  is  a  hard  nodule  beneath  the  skin  of  the  thorax 
On  mcision  this  is  found  to  consist  chiefly  of  tough  whitish  tissue,  evidently  of  the 
nature  of  a  scar,  and  presents  nothing  suggestive  of  a  verruga  lesion.  The"' surface 
of  both  testes  shows  red  spots  about  25  to  30  in  number  from  mere  points  to  3  mm. 
m  diameter.  On  opening  the  sac  these  are  found  to  be  rather  in  the  sac  than  in  the 
testes  proper.  On  close  inspection  these  appear  elevated  shghtly  above  surface 
and  consist  of  flattened  areas  with  rounded  outline  surrounded  by  dehcate  blood- 
vessels. The  interior  of  left  testis  presents  no  lesion.  Axillary  lymph-nodes  shghtly 
enlarged,  but  greatest  on  side  opposite  to  lesion.  The  left  portion  of  the  omentum 
is  adherent  around  the  spleen  and  shows  a  great  number  of  small  red  rounded 
lesions  from  mere  points  to  2  mm.  in  diameter.  In  the  region  of  this  a  small  area 
at  the  edge  of  the  spleen  is  of  a  grayish  color;  it  is  evidently  necrotic,  measures 
2X4  mm.     Other  organs  appear  normal, 

Rahbit  No.  6^.  —  Culture  inoculation  into  testes. 

July  30,  1913.     Both  testes  inoculated  with  0.5  c.c.  culture  of  virus  from  Case  19. 
Augu.st    3.    Testes  normal. 

August  28.     Killed.     Both  testes  appear  normal.     All  other  organs  appear 
normal.    No  tissue  saved. 


118  VERRUGA  PERUVIANA- 

Rabbit  No.  66.  —  Testes  inoculation,  2d  transfer. 

August  3,  1913.  Both  testes  inoculated  with  suspension  of  lesion  from  Rabbit 
No.  52. 

August  11.  Found  dead.  Rigor.  Both  testes  present  lesions  from  1  mm.  to 
3  or  4  mm.  opaque  gray  with  yellow  tinge,  evidently  necrotic  in  central  portion. 
Fixed  in  formol.  Smear  stained  by  Wright's  stain  shows  a  small  number  of  large 
bacilli  with  red  stained  centers.  Cells  loaded  with  granules,  deeply  stained  and 
of  varjdng  size. 

Rabbit  No.  67.  —  Testes  inoculation,  2d  transfer. 

August  3,  1913.  Both  testes  inoculated  with  suspension  of  lesion  from  Rabbit 
No.  52. 

August  11.  Found  dead.  Considerable  post  mortem  decomposition.  Testis 
dark  reddish  with  numerous  lesions  appearing  as  grayish  opaque  spots  2  to  3  mm. 
Testis  in  formol. 

Rabbit  No.  68.  —  Testes  inoculation,  2d  transfer. 

August  3,  1913.  Both  testes  inoculated  with  suspension  of  lesion  from  Rabbit 
No.  52. 

September  16.  Left  side  of  scrotum  is  large  and  firm  throughout.  The  testis  is 
scarcely  discernible  in  this  mass.  The  right  testis  is  somewhat  swollen  but  presents 
no  distinct  nodules. 

September  17.  Found  dead.  Belly  somewhat  distended  and  considerable 
post  mortem  discoloration.  The  entire  testis  replaced  bj"^  inflammatory  tissue 
showing  extensive  necrosis.  The  necrotic  portions  are  firm,  yellowish  white,  and 
subdivided  into  alveoli.  The  right  testis  shows  a  large  abscess  at  the  posterior 
extremity  containing  thick  yellowish  puriform  material.  A  small  yellowish  spot 
is  seen  in  the  anterior  extremity.  This  also  consists  of  puriform  material.  Post 
mortem  decomposition  rather  marked.     No  tissue  saved. 

Rabbit  No.  71 .  —  Testes  inoculation,  2d  transfer. 

August  8,  1913.  Both  testes  inoculated  with  lesion  of  Rabbit  No.  53,  ground  in 
salt  solution. 

August  28.  Killed.  One  testis  shows  a  lesion  about  3  mm.  in  diameter  with 
slightly  yellowish  tinge  and  opaque  white  in  center.  Nodule  is  of  firm  consistency 
and  bulges  slightly  from  the  cut  surface.  Nodule  in  other  testis  is  somewhat  larger, 
about  3.5  mm.  in  diameter,  and  of  similar  appearance.  Nodules  ground  in  salt 
solution  and  used  to  inoculate  four  rabbits  Nos.  82  to  85,  and  one  monkey  No.  67. 
No  tissue  saved. 

Rabbit  No.  72.  —  Testes  inoculation,  2d  transfer. 

August  8,  1913.  Both  testes  inoculated  with  lesion  of  Rabbit  No.  53,  ground 
in  salt  solution. 

September  16.  Right  testis  presents  a  minute  hard  nodule,  the  left  an  area 
3  to  4  mm.  in  diameter.     Scrotum  normal. 

September  19.  Killed.  Right  testis  is  small  and  shows  a  spot  on  ventral 
surface  with  a  small  elevated  area.  On  section  there  is  a  larger  area  within  its 
substance  measuring  approximately  4  mm.,  and  consisting  of  an  aggregation  of 
smaller  lobules  each  of  which  shows  a  whitish  interior.  The  left  testis  shows  a 
nodule  in  its  interior  measuring  7  X  4.5  mm.  On  section  it  shows  a  considerable 
portion  of  whitish  tissue  evidently  necrotic.  Some  of  this  is  easily  scraped  away 
and  is  rather  soft.  Near  the  periphery  the  tissue  is  translucent  with  a  faint  yellow- 
ish tinge.     Two  cultures  made  from  left  testis.     Half  fixed  in  Zenker's  solution,  the 


EXPERIMENTS  119 

remainder  used  for  inoculation  of  two  rabbits,  Numbers  92  and  93.  The  other 
organs  show  no  lesions  of  importance.  Rabbit  was  in  rather  poor  condition,  some- 
what emaciated. 

September  26.  Two  cultures  from  the  left  testis,  both  remained  sterile  after 
seven  days. 

Experiments  with  Case  21 

Clinical  data:  Male,  adult,  observed  at  San  Bartolome,  July  23,  1913. 
Patient  has  been  living  in  San  Bartolome  for  a  month  or  more  working 
on  the  railway.  There  is  a  profuse  papular  eruption  over  both  surfaces 
of  the  forearms,  and  backs  of  hands  and  over  the  legs.  The  papules 
measure  from  3  to  5  mm.  in  diameter,  are  pink  in  color,  sharply  circum- 
scribed, firm  to  the  touch,  and  appear  to  be  in  a  moderately  active  stage. 
The  majority'  of  them  are  flattened  on  the  surface,  and  not  dome-shaped, 
being  raised  for  a  distance  of  1  or  2  mm.  above  the  surface  of  the  skin. 
Patient  states  eruption  has  been  out  about  twelve  days,  and  that  he  had 
slight  fever  and  pain  in  the  joints  for  one  or  two  days  before  it  appeared. 
Three  of  the  papules  were  removed  from  the  skin  after  it  had  been  disin- 
fected with  alcohol,  ether,  and  iodin.  One  was  placed  in  Zenker's  solution, 
one  used  for  antigen  and  a  third  one  for  the  inoculation  of  a  monkey 
as  follows: 

Inoculation  of  Animals 

Monkey  No.  3.  —  Cutaneous  inoculation. 

August  1,  1913.  At  camp  on  mountain  above  San  Bartolom<5.  Inoculated 
monkey  on  brows,  tip  of  nose,  inner  and  outer  surface  of  lower  Up  and  belly  by 
scarification.     A  small  implant  made  by  needle  in  posterior  surface  of  each  ear. 

August  18.  Right  eyelid  shows  linear  reddened  elevation.  Reddish  papule 
on  left  brow.     Ear  shows  a  firm  mass. 

August  21.     Reddened  elevations  more  distinct,  now  a  papule  on  both  brows. 

August  23.  Examination  complete  except  inner  surface  of  lip.  Eyelid,  both 
brows,  nose  and  lower  lip  show  lesions.  These  are  reddish  papules  elevating  the 
surface,  the  largest  not  over  2  or  3  mm.  Those  on  right  brow,  nose  and  lip  have 
been  excoriated  intensifying  the  reddening,  making  lesions  appear  more  striking. 
Over  left  thorax  is  a  lesion  of  the  size  of  a  split  pea,  similar  nodule  in  each  ear. 

August  25.  Papules  on  brows  somewhat  increased  in  size,  quite  red,  that  on 
nose  has  become  somewhat  rounded  2.5  to  3  mm.,  and  is  bright  red.  The  lower 
lip  now  shows  nothing  definite  externally. 

August  28.  Lesion  on  left  eyebrow  excised,  and  used  for  inoculation  of  four 
rabbits  Nos.  86  to  89,  and  two  monkeys  Nos.  8  and  9  (q.  v.).  January  22,  eyebrows 
normal. 

Monkey  No.  8.  —  Inoculated  subcutaneously. 

August  28,  1913.  Inoculated  on  both  eyebrows,  tip  of  nose  and  right  thorax 
with  suspension  of  lesion  taken  from  thorax  of  Monkey  No.  3,  ground  in  salt  solu- 
tion. 

September  16.  Cough  has  been  present  and  increasing  for  at  least  a  week. 
Considerable  emaciation;  animal  appears  pale  and  weak. 

September  18.  About  0.5  cm.  beneath  the  nipple  is  a  rounded  nodule  5X4 
X  2  mm.  approximately. 


120  VERRUGA  PERUVIANA 

September  20.  Found  dead.  Small  nodule  about  1  cm.  below  right  nipple 
situated  subcutaneously.  This  measures  about  5  X  3.5  X  2  mm.  and  has  the 
appearance  of  an  abscess,  being  grajdsh  white.  Nose  and  eyebrows  appear  normal. 
Left  axillary  lymph-nodes  are  considerably  enlarged  and  an  abscess  containing 
cheesy  material  the  size  of  a  mustard  seed  is  found.  The  lower  lobe  and  about 
one-fourth  of  the  upper  lobe  of  the  left  lung  is  consohdated.  Near  the  root  of 
the  lower  lobe  is  an  abscess  measuring  1.5  cm.,  and  there  are  numerous  small 
abscesses  throughout  this  lobe.  These  are  yellowish  or  grayish  in  color,  the  larger 
ones  containing  viscid  puriform  material.  In  the  consohdated  portion  of  the  upper 
lobe  are  yellowish  abscesses,  some  discrete,  others  confluent.  There  are  scattered 
lesions  throughout  the  other  portions  of  the  lungs.  These  are  rather  translucent 
and  grayish  with  a  more  opaque  center.  Throughout  the  right  lung  are  small 
nodules  measuring  from  1  to  3  mm.  These  are  translucent  at  the  periphery  and 
opaque  whitish  gray  in  the  center.  There  is  one  abscess  containing  whitish  caseous 
material.  Attached  to  the  rib  on  the  left  thorax  is  an  abscess  about  0.8  cm.  in 
diameter,  and  one  of  similar  size  and  appearance  in  the  lower  aspect  of  the  liver, 
left  central  lobe,  and  scattered  abscesses  throughout  the  omentum  and  spleen. 
Pericardium  is  smooth,  shows  no  exudate.  There  is  an  abscess  in  the  wall  of  the 
right  ventricle  and  around  the  pulmonary  artery.     Tissue  in  Zenker's  solution. 

Monkey  No.  9.  —  Cutaneous  inoculation. 

August  28,  1913.  Inoculated  on  brows  and  tip  of  nose  by  scarification  with 
lesion  taken  from  eyebrow  of  Monkey  No.  3. 

September  16.  Reddish  rounded  papules  on  brows  measuring  approximately 
3  to  4  mm.,  and  elevated  2  mm.  These  were  well  developed  when  seen  yesterday 
and  are  apparently  slightly  larger  today. 

September  18. 
Lesion  A,  measures  6X5X4  mm.,  dark  cherry,  thin  crust  on  surface. 
«        B,         "  3  X  3  X  2.5  mm.  scraped  off  surface. 

"        C,         "  2.5  X  2.5  X  2.5  mm.  conical. 

«        D,        "  9X3X2  nam.  confluent. 

September  19.  Lesion  "  C  "  excised  and  placed  in  Zenker's  solution.  Lesion 
"  D  "  excised  and  used  for  inoculation  purposes  (Rabbits  Nos.  94  to  97,  Monkeys 
Nos.  11  and  12,  and  Dog  No.  6  see  Tables  i  and  ii).  The  crust  removed  from 
lesion  "  A  "  and  bits  scraped  oU  for  the  inoculation  of  Monkeys  Nos.  10  and  11. 

September  27.  Lesion  "  A  "  presents  a  red  granulating  surface  and  is  about 
the  same  size  or  has  shghtly  increased;  it  appears  now  as  before  the  operation. 
Lesion  "  B  "  shows  shght  change  and  is  surmounted  by  a  crust.  There  is  an 
elevated  area  surmounted  by  a  crust  at  site  of  lesion  "  C."  Lesion  "  D  "  is  now 
replaced  by  a  smooth  scar. 

September  29.  Lesion  "  A  "  somewhat  increased  in  size,  approximately  spheri- 
cal with  red  granulating  surface.     Lesions  "  B  "  and  "  C  "  also  slightly  increased. 

October  17.     Eyebrows  appear  normal. 

December  3.  Dead.  Noticed  sickly  about  a  week  ago.  Autopsy  immediately 
following  death.  Peristalsis  observed  in  intestines.  The  lower  lobe  of  the  right 
lung  is  consolidated,  also  the  greater  part  of  the  middle,  and  portions  of  the  upper 
lobe.  On  section  there  are  small  nodules  with  dry  grayish  caseous  material  and 
abscess  cavities  containing  shmy  mucopurulent  material.  A  smear  made  from  the 
latter  shows  several  acid-fast  baciUi.  The  rectal  mucus  examined  for  Balantidium 
coll  but  none  are  present,  although  there  are  a  few  amoebae  present  in  the  mucus. 
There  are  no  ulcerations.  Spleen  small;  other  organs  show  no  lesions.  Cultures 
on  agar  and  bouillon.     No  tissue  saved. 


EXPERIMENTS  121 

Rabbit  No.  86.  —  Testes  inoculation. 

August  28,  1913.  Both  testes  inoculated  with  0.5  c.c.  suspension  of  lesion  from 
thorax  of  Monkey  No.  3,  ground  in  salt  solution. 

September  12.  Found  dead.  No  lesion  seen  in  either  testis,  both  placed  in 
Zenker's  solution.     Other  organs  appear  normal. 

Rabbit  No.  87.  —  Testes  inoculation. 

August  28,  1913.  Both  testes  inoculated  with  0.5  c.c.  suspension  of  lesion  from 
thorax  of  Monkey  No.  3,  ground  in  salt  solution. 

September  16.     Both  testes  show  nodules,  left  larger,  not  over  2  or  3  mm. 

September  17.  Dead.  Right  testis  shows  a  small  vascular  elevation  with  a 
whitish  center  about  1  mm.  in  diameter.  In  the  interior  is  a  nodule  measuring 
2  mm.  in  diameter,  of  firm  consistence  and  made  up  of  an  aggregation  of  small, 
opaque,  whitish  lobules  with  a  slight  yellowish  tinge.  The  left  testis  shows  a 
similar  lesion  just  beneath  the  surface.  In  the  interior  is  a  similar  nodule  with 
less  necrosis  as  the  periphery  appears  somewhat  translucent.  The  peritoneal  sur- 
face is  smooth.  The  wall  of  the  small  intestine  shows  a  moderate  number  of 
opacities  possibly  coccidial  lesions.  The  liver  shows  several  characteristic  coccidium 
nodules.     Lungs  are  normal.     Tissue  in  Zenker's  solution. 

Rabbit  No.  88.  —  Testes  inoculation. 

August  28,  1913.  Both  testes  inoculated  with  0.5  c.c.  suspension  of  lesion  from 
thorax  of  Monkej'^  No.  3,  ground  in  salt  solution. 

September  16.  Right  small  nodule  within  testis,  also  two  firm  nodules  beneath 
skin,  left  fairly  large  nodule  anterior  end  of  testis. 

September  30.  Killed.  Right  testis  shows  a  small  lesion  occupying  about  ^ 
of  the  testis  consisting  of  scattered  irregular  nodules  without  necrosis  and  of  slightly 
yellowish  color.  Whitish  opalescent  nodule  bulging  from  the  surface.  Left  testis 
shows  a  small  lesion  in  the  interior.  Other  organs  appear  normal.  Tissue  in 
Zenker's  solution. 

Rabbit  No.  89.  —  Testes  inoculation. 

August  28,  1913.  Both  testes  inoculated  with  0.5  c.c.  suspension  of  lesion  from 
eyebrow  of  Monkey  No.  3,  ground  in  salt  solution. 

September  16.     Testes  very  small,  left  shows  a  distinct,  hard  nodule. 
October  17.     Both  testes  negative. 
December  5.     Rabbit  normal. 

Experiment  with  Case  25 

August  2,  1913.  The  patient  is  an  Englishman,  who  has  been  working 
in  the  mines,  and  has  slept  for  three  nights  in  the  verruga  district  near 
Matucana.  He  was  perfectly  well  up  to  June  1,  1913.  Then  he  began 
to  feel  slightly  ill.  At  this  time  he  noticed  a  small  red  nodule  below  the 
right  knee.  This  developed  gradually  in  size.  In  the  course  of  a  week  a 
second  nodule  developed  below  the  right  knee,  and  two  weeks  after  the 
appearance  of  the  first  a  third  developed  below  the  left  knee.  The  patient 
states  that  he  had  fever  at  this  time  lasting  for  about  ten  days,  and  accom- 
panied by  pains  in  his  joints  and  head  and  back.  The  fever  left  him  after 
about  ten  days.  The  nodules  have  persisted  up  to  the  present  time,  having 
remained  stationary  for  about  the  past  two  or  three  weeks.      At  present 


122  VERRUGA  PERUVIANA 

there  are  three  cherry  red  nodules,  two  below  the  right  knee,  measuring 
about  5  to  7  mm.  in  diameter,  and  one  below  the  left  knee  measuring 
about  8  mm.  in  diameter.  They  are  raised  above  the  surrounding  skin. 
The  one  on  the  left  leg  was  removed  with  scissors,  and  a  monkey  inoculated 
as  follows: 

Monkey  No.  4.  —  Cutaneous  inoculation. 

August  2,  1913.  Inoculated,  with  portion  of  lesion  removed  from  patient,  right 
side  of  abdomen,  face,  eyebrow,  and  eyelid  by  scarification;  right  breast  and  pos- 
terior surface  of  right  ear  twice  by  implant. 

August  3.  Animal  noted  with  eyes  rolling  and  arms  twitching,  and  died  soon 
afterward. 

Transmission  of  the  Virus  of  Verruga  to  Animals 

From  the  experiments  previously  recorded  in  this  chapter  in 
regard  to  the  transmission  of  the  virus  to  animals,  and  from 
Tables  i  and  ii,  it  may  be  seen  that  the  monkey  is  the  most 
satisfactory  animal  for  inoculation  purposes.  If  the  skin  over 
the  eye  is  scarified  and  a  small  portion  of  a  verruga  nodule 
rubbed  into  the  abrasions,  after  an  incubation  period  usually 
of  ten  to  twenty  days,  small  papules  appear  which  gradually 
enlarge  and  later  assume  the  typical  picture  of  the  verruga 
nodules  as  seen  in  human  beings.  (Plates  xxiv  and  xxv.)  The 
nodules  thus  produced  also  have  a  similar  histological  structure 
to  those  observed  in  man,  a  fact  that  will  be  referred  to  again. 
We  have  transmitted  this  virus  from  animal  to  animal  through 
twelve  successive  series  of  monkeys  since  our  departure  from 
South  America  over  a  year  ago.  In  thirty-four  monkeys 
typical  lesions  have  been  produced  in  this  manner. 

When  the  monkey  is  inoculated  with  the  virus  either  directly 
from  man  or  from  another  monkey,  no  generalized  eruption 
occurs;  as  in  the  case  when  this  animal  is  inoculated  with 
small-pox  virus  onl}^  a  modified  form  of  the  disease  develops. 
It  is  probable  that  the  monkey  is  not  as  susceptible  to  the 
virus  of  verruga  or  even  to  that  of  small-pox  as  is  man.  The 
virus  of  verruga  also  calls  forth  no  appreciable  febrile  reaction 
in  the  monkey  inoculated  with  it,  and  none  of  the  monkeys 
died  from  the  inoculation  with  this  virus.  The  lesion  in  the 
monkey  usually  begins  to  regress  four  or  five  weeks  from  the 
time  of  the  inoculation. 


A  ,# 


/^ 


4. 


"^0^ 


^^^ 


Plate  XXIV  — Verruga  Inoculata  in  the  Monkey 


p 

jiri 

■  '^-       '■^t 

H^^^yL^-^ 

^^^^flK 

■^f^>^Hlp'    " 

T-'^'^^W 

^I^^E^^            i 

|| 

R 

^^^^m/ 

1 

Plate  XXV.  —  Verruga  Pehuviana.     Expeririiental  lesions  produ(!od  in  monkeys 
bv  inoculation  of  human  virus. 


TABLE   I.     ANIIMAL  INOCULATIONS 


With  ^Material  from  ^'ERRUGA  Case  1 


Animal  No. 

Rabbit  1 
"  2 
3 
4 
5 
6 
7 


Transfer 


Site  of  Inoculation 


Result 


Dog 


Testis 

+ 

II 

+ 

II 

+ 

Cornea 

— 

Intraperitoneally 

— 

Cutaneously  and 

Subcutaneously 

— 

Siibcutaneously  and 

Intraperitoneally 

+ 

Testis 

— 

a 

+ 

u 

— 

a 

+ 

.bbi 

it    8 

« 

Testis 

II 

9 

II 

a 

II 

10 

" 

u 

II 

11 

II 

a 

II 

12 

II 

« 

II 

13 

II 

Testis  with  Filtrate 

II 

14 

u 

II         II          II 

II 

15 

II 

Testis 

i< 

16 

II 

Intraperitoneally 

II 

17 

II 

"      with  Filtrate 

II 

28 

II  Rabbit 

2 

Testis 

u 

29 

II 

II 

2 

II 

u 

30 

« 

II 

2 

« 

u 

31 

II 

II 

2 

II 

u 

32 

II 

« 

2 

" 

a 

44 

II 

<i 

3 

« 

u 

45 

II 

II 

3 

II 

u 

46 

" 

II 

3 

" 

•g 

2 

II 

Dog 

1 

II 

II 

3 

" 

" 

1 

Cutaneously 

bbi 

It  47 

II 

" 

1 

Testis 

" 

54 

II 

Rabbit 

9 

II 

u 

55 

II 

II 

10 

" 

« 

56 

II 

II 

11 

II 

II 

57 

III 

« 

46 

" 

u 

58 

II 

11 

46 

11 

u 

59 

" 

II 

46 

II 

u 

60 

" 

« 

46 

II 

" 

61 

Cultures 

II 

" 

62 

« 

u 

« 

69 

III  Rabbit  55 

II 

" 

70 

" 

Dog 

2 

" 

" 

73 

II  Human 

I  vaccina 

- 

tion 

II    . 

+ 


+ 


+ 
+ 
+ 
+ 

+ 

+ 


+ 


Killed 
After  Inocul'n 

38  days 
17  « 

24  « 

28  " 

28  " 

25  " 

37  « 

26  " 
26  " 
34  " 
34  « 
26  " 

26  " 

27  « 

39  « 

28  « 
28  " 

28  " 
32  « 
32  « 

32  « 

33  « 

32  « 
25  « 
25  « 
67  « 
23  " 
23  « 
45  " 
17  « 
17  " 
12  « 
30  " 

33  " 
33  " 
62  " 

29  « 
29  " 
53  " 
19  « 

25  " 


Killed 

Animal  No. 

Transfer 

Site  of  Inoculation 

Result 

After  Inoculation 

Rabbit  74 

III  Rabbit  31 

Testis 

— 

16  days 

"       75 

a 

ii 

31 

(t 

—  (septic) 

52     « 

"       76 

u 

a 

31 

(1 

— 

40     " 

"       77 

u 

« 

31 

il 

+ 

52     " 

«       78 

IV 

a 

57 

a 

—  (septic) 

33     " 

"       79 

u 

a 

57 

a 

— 

a 

29     " 

«       80 

ii 

it 

57 

a 

— 

a 

33     " 

«       81 

u 

a 

57 

« 

— 

13     " 

Monkey  6 

11 

ii 

57 

Subcutaneously 

— 

Rabbit  90 

III 

" 

46 

Testis 

— 

6     " 

"       91 

u 

(1 

46 

a 

— 

28     " 

"       98 

IV 

a 

77 

ii 

•  —  (septic) 

17     " 

"       99 

il 

a 

77 

a 

— 

a 

17     " 

"With  Material  from  Verruga  Case  7 

Rabbit  24 

I 

Testis 

— 

28  davs 

«       25 

a 

a 

— 

33     « 

«       26 

u 

It 

— 

33     « 

"       27 

u 

» 

— 

33     " 

"       65 

II  Rabbit  24 

<( 

— 

53     « 

With  Blood 

FROM  Case  8  (Oroya 

Fever) 

Rabbit  20 

Testis 

— 

22  days 

«       21 

a 

— 

28     « 

«       22 

u 

— 

24     " 

"       23 

Intravenously 

— 

23     " 

With  Blood 

from  Case  15  (Oroya 

Fever) 

Rabbit  33 

Testis 

— 

28  days 

«       34 

u 

— 

2     « 

"       35 

u 

— 

28     « 

«       36 

Intravenously 

— 

28     " 

Monkey  1 

» 

— 

39     « 

With  Material  from  Verruga  C 

ASE    16 

Rabbit  37 

I 

Testis 

— 

10  days 

"       38 

u 

a 

— 

2     " 

"       73 

II 

• 

ii 

+ 

25     " 

Monkey  3 

I 

Cutaneously 

+ 

20     " 

With  Material  from  Verruga  Case  17 

Rabbit      39 

I 

Testis 

— 

20  days 

40 

it 

Intravenously 

— 

11     " 

41 

u 

Intraperitoneally 

— 

29     " 

42 

ii 

Testis 

— 

29     " 

43 

a 

a 

— 

9     " 

Guinea  Pig  1 

u 

Intraperitoneally 

— 

16     " 

"            "     9 

a 

« 

— 

13     " 

Monkey      2 

a 

Cutaneously 

— 

Rabbit      63 

Cultures 

Testis 

— 

29     « 

With  ]\Iaterial  from  Verruga  Case  19 


Animal  Xo. 

Fransfer 

Site  of  Inoculation 

Result 

KUled 
After  Inoculation 

Rabbit  48 

I 

Testis  with  Filtrate 

— 

22  days 

"       49 

« 

U                    U                      il 

— 

42     « 

"       50 

u 

a              u               it 

— 

22     « 

51 

u 

it              il               » 

— 

42     " 

"       52 

u 

Testis 

+ 

17     " 

"       53 

u 

u 

+ 

44     " 

Dog        4 

u 

Testis  with  Filtrate 

— 

23     " 

"         5 

" 

"    and  Subcutaneously 

+ 

23     « 

Rabbit  64 

Cultures 

Testis 

_ 

29     « 

«       66 

II  Rabbit  52 

(( 

-  (septic)       8     " 

"       67 

u 

u 

52 

« 

— 

8     " 

"       68 

u 

u 

52 

tt 

— 

41     " 

«       71 

u 

u 

53 

tt 

+ 

20     " 

"       72 

" 

u 

53 

ti 

+ 

42     " 

"       82 

III 

u 

71 

(1 

+ 

33     « 

«       83 

<( 

" 

71 

(( 

— 

33     « 

"       84 

(( 

(1 

71 

(( 

— 

16     « 

«       85 

u 

(1 

71 

it 

— 

18     « 

92 

ti 

u 

72 

it 

— 

28     " 

"       93 

u 

u 

72 

It 

— 

11     " 

«     100 

IV 

u 

82 

" 

+ 

17     " 

With  Material  from  Verruga  Case 

21 

Lesions  Present 

Animal  No. 

Transfer 

Site  of  Inoculation 

Result 

After  Inocul'n 

Monkey    3 

I 

Cutaneously 

+ 

27  days 

Rabbit    86 

II : 

Monkey 

Testis 

— 

87 

" 

(1 

it 

+ 

20     " 

88 

u 

u 

a 

+ 

19     " 

89 

u 

u 

u 

— 

Monkey    8 

u 

il 

Cutaneously  and  Sub- 
cutaneously 

— 

9 

it 

" 

((         It         It 

+ 

18  to  53  days 

Rabbit    94 

III 

(( 

Testis 

+ 

28  days 

95 

" 

u 

It 

+ 

28     " 

96 

li 

u 

u 

+ 

28     " 

97 

" 

u 

It 

+ 

28     « 

Monkey  10 

u 

Cutaneously  and  Sub- 
cutaneously 

+ 

8  to  28  days 

11 

u 

" 

(1        it        it 

+ 

10  "  28     " 

12 

u 

(( 

It        It        It 

+ 

8  "  28     " 

Dog          6 

u 

u 

Subcutaneously 

+ 

11  "  25     « 

Monkey  13 

IV 

u 

Filtrate 

Cutaneously 

— 

14 

" 

" 

u 

« 

— 

15 

« 

<( 

u 

u 

— 

16 

u 

it 

Virus 

u 

+ 

17 

u 

u 

u 

u 

+ 

With  Material  from  Verruga  Case 

24 

Animal  No. 

Transfer 

Site  of  Inoculation                  Result 

Died 
After  Inocul'n 

Monkey  4 

I 

Subcutaneously  and 

Cutaneously 


2  days 


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TRANSMISSION   OF  VIRUS   TO  ANIMALS  123 

From  the  experiments  recorded  in  this  section,  and  from 
Table  I,  It  may  also  be  seen  that  the  virus  may  be  sometimes 
transmitted  to  rabbits  and  to  dogs.     In  the  dog  the  subcu- 
taneous moculation  occasionally  produces  a  lesion  which  is 
much  more  vascular  than  the  one  produced  in  the  rabbit's 
testis.     Upon  intratesticular  inoculation  of  the  virus  into  rab- 
bits or  dogs,  a  more  or  less  characteristic  lesion  is  sometimes 
produced  after  an  incubation  period  varying  usually  from  ten 
to  twenty-two  days.     (Plate  xxvi.)     The  lesion  usually  meas- 
ures several  millimeters  in  diameter,  and  is  sharply  circum- 
scribed from  the  surrounding  tissue.     In  the  rabbit's  testis  it 
suggests  m  its  appearance  not  a  lesion  which  has   resulted 
from  an  acute  inflammatory  process,  but  rather  one  which  has 
arisen  from  a  rather  low  grade  of  inflammation.     By  repeated 
inoculations  of  this  character,  however,  the  virus  becomes  grad- 
ually attenuated,  and  finally  inoculations  of  this  nature  fail.    In 
only  a  certain  percentage  of  the  inoculated  rabbits  does  a  lesion 
develop,  as  may  be  seen  from  Table  i.    This  fact  seems  to  show 
that  rabbits  are  not  very  susceptible  to  the  virus. 

In  the  earlier  literature  upon  the  subject  of  verruga  peru- 
viana and  Oroya  fever,  it  has  been  repeatedly  stated  that  the 
lower  animals  were  susceptible  to  the  disease,  and  that  the 
infection  had  been  observed  in  dogs,  cats,  mules,  and  poultry. 
Scheube  and   Manson  refer  to   these  statements.     Manson 
points  out  in  this  connection  that  if  the  lower  animals  were 
susceptible  to  the  disease,  it  is  curious  that  Carrion  chose  to 
experiment  upon  himself.     Scheube  calls  attention  to  the  fact 
that  animals  have  also  been  reported  to  suffer  from  Oroya 
fever.     Barton    claimed    the    production    of    a   verruga-like 
eruption  upon  the  skin  of  dogs  following  the  inoculation  of  an 
organism  said  to  have  been  cultivated  from  cases  of  Carrion's 
fever,  and  afterwards  shown  to  be  a  paracolon  bacillus.     As  a 
matter  of  fact  there  was  no  demonstration  that  the  virus  of 
verruga  could  be  transmitted  to  animals  until  experiments 
with   monkeys   were   undertaken.     Jadassohn   and   Seiffert  ^ 

^^^  Jadassohn  and  Seiffert:  Ztschr.  f.  Hyg.  u.  Infectionskrankh,  Lcipz.,  1910,  Ixvi, 


124  VERRUGA  PERUVIANA 

in  one  case  of  verruga  peruviana,  and  Mayer,  Rocha-Lima, 
and  Werner  ^  in  another  case,  both  of  which  were  studied  in 
Europe,  also  succeeded  in  transmitting  the  virus  to  monkeys 
through  several  generations.  Cole  ^  showed  from  a  study  of 
Jadassohn's  case,  and  the  material  from  the  animal  lesions, 
that  the  histological  structure  of  the  nodules  produced  in  the 
monkeys  was  also  similar  to  that  of  the  human  lesions.  In 
Lima  more  recently  Dr.  Ribeyro  also  transmitted  the  virus 
of  verruga  to  a  monkey  in  which  we  observed  the  lesions 
develop.  So  far  as  we  know  up  to  the  time  of  writing  no 
publication  of  this  experiment  has  yet  been  made.  ^  These 
experiments  and  our  own  upon  monkeys,  dogs,  and  rabbits, 
already  described,  are  the  only  ones  that  have  demonstrated 
that  the  virus  of  verruga  is  transmissible  to  the  lower  animals. 
We  did  not  observe  any  cases  of  natural  infection  of  the  disease 
in  dogs,  cats,  donkeys,  or  poultry,  and  do  not  believe  the 
disease  occurs  naturally  in  these  animals.  In  fact  the  monkey 
is  the  only  really  satisfactory  animal  for  inoculation  purposes 
in  this  disease.  In  the  transmission  of  the  virus  from  man  to 
animals  it  is  noticeable  that  in  the  monkey,  and  as  has  been 
mentioned  particularly  in  the  intratesticular  inoculation  of  the 
rabbit,  the  inoculation  often  fails,  and  no  lesion  results.  This 
may  be  seen  from  Tables  i  and  ii,  and  is  particularly  striking 
in  the  experiments  performed  with  Cases  1,  7,  17,  19,  and  21. 
It  would  appear  that  the  virus,  even  in  man,  is  only  transmis- 
sible during  a  certain  stage  of  the  disease,  and  that  unless  it  is 
obtained  from  the  lesion  of  the  human  case  during  that  stage, 
it  is  either  innocuous  for  animals  or  its  virulence  is  greatly 
reduced.  Also,  the  susceptibility  of  the  animal  to  the  infec- 
tion obviously  must  play  a  role  in  regard  to  the  percentage  of 
animals  in  which  the  inoculation  succeeds  and  a  definite 
lesion  results.  In  monkeys  the  percentage  of  successful 
inoculations  is  much  higher  than  in  rabbits,  and  the  virus, 
while  it  sometimes  becomes  attenuated  by  successive  passages 
through  these  animals,  apparently  usually  does  not  become 

^  Mayer,  Rocha-Lima,  Werner:  loc.  cit. 

2  Cole:  J.  Cutan.  Dis.,  N.Y.,  June,  1913,  xxxi,  384. 

'  This  experiment  has  since  been  published  Trop.  Dis.  Bull.,  Lond.,  1914,  iv,  486. 


B 

1 

i^"H 

Fig.  1.  —  Dog's  testis.    Showing  experimental  lesion  produced 
by  the  inoculation  of  virus  from  a  human  l)eing. 


1 

1 

1 

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I'id.  2.  —  IIahdit's  tkstis.     Shovvuifi  lesion  pioduccd  by  the 
inoculation  of  human  virus. 

Plate  XXVI.  —  Vekkuga  Peruviana. 


CULTIVATION   AND   FILTERABILITY   OF   VIRUS       125 

attenuated  to  the  same  degree  as  it  does  by  successive  passages 
through  the  rabbit's  testis.  When  once  the  virus  has  been 
successfully  transmitted  from  the  human  lesion  to  the  monkey, 
it  may  be  carried  through  many  successive  passages  in  these 
animals,  as  is  shown  in  Table  ii. 

Cultivation  and  Filterability  of  the  Virus  of  Verruga 

In  many  of  the  experiments  already  referred  to  in  this  sec- 
tion the  attempts  at  cultivation  of  the  virus  of  verruga  from 
material  obtained  from  the  human  lesions  and  from  those 
produced  in  the  rabbit's  testis  have  been  considered,  and  the 
results  recorded.  From  these  experiments  it  may  be  seen 
that  even  in  those  cultures,  which  suggested  from  their  ap- 
pearance the  possibihty  of  a  growth,  no  definite  microorganism 
could  be  demonstrated  by  microscopical  examination  or  by 
subculture,  and  upon  inoculation  of  some  of  these  cultures  into 
rabbits'  testes  no  lesions  were  produced.  While  the  results 
alone  of  the  inoculation  experiments  into  animals  of  this  nature 
do  not  preclude  a  cultivation  of  the  virus,  since  by  this  method 
of  inoculation  as  we  have  seen  from  Table  i  only  a  small 
proportion  of  rabbits  may  be  expected  to  develop  lesions  even 
when  the  fresh  virus  is  employed,  nevertheless,  the  results  of 
these  experiments  do  not  give  any  support  to  the  idea  of  a 
successful  cultivation  of  the  virus.  Numerous  attempts  have 
also  been  made  to  cultivate  the  virus  in  vitro  from  the  verruga 
lesions  produced  in  monkeys  by  its  inoculation.  These 
experiments  like  those  already  considered  in  relation  to  the 
cultivation  of  the  virus  from  the  human  and  rabbit  lesions 
have  usually  resulted  in  failure.  Various  modifications  of 
the  methods  described  by  Noguchi  and  Murphy  for  cultiva- 
tion have  been  employed.  Great  difficulty  has  been  experi- 
enced in  obtaining  the  material  from  monkeys  free  from 
bacteria.  The  monkeys  almost  invariably  scratch  and  infect 
secondarily  the  lesions.  It  is  not  deemed  advisable  to  con- 
sider in  detail  all  of  the  experiments  which  have  resulted  in 
complete  failure  of  cultivation  of  the  virus  from  the  lesions 
produced  in   these   animals.     However,   the  results  will   be 


126  VERRUGA  PERUVIANA 

briefly  referred  to  which  were  observed  in  a  series  of  cultures 
made  in  ascitic  fluid  containing  rabbit's  testis  to  which  had 
been  added  exudates  produced  by  injections  of  aleuronat 
containing  leukocytes,  and  small  fragments  of  verruga  nodules. 
In  several  instances  such  cultures  showed  certain  differences 
from  the  control  tubes,  notably  in  a  cloudiness  of  the  media, 
and  in  the  formation  of  a  flne  granular  precipitate  along  the 
sides  and  bottom  of  the  tube.  This  precipitate  perhaps 
resulted  from  disintegration  of  the  tissues  in  the  culture  by  the 
growth  of  the  virus.  Apparently  it  did  not  constitute  a  true 
culture  as  examination  revealed  no  bacteria  or  other  visible 
microorganism,  and  subcultures  on  agar  gave  no  apparent 
growth.  In  the  case  of  one  monkey,  see  Table  ii,  inoculated 
with  such  a  culture  which  had  been  incubated  for  sixteen  days 
at  37°  C,  a  small  group  of  reddish  papules  appeared  in  a  row 
on  each  brow  after  an  incubation  period  of  twenty-one  days. 
These  papules,  however,  never  developed  into  as  extensive 
lesions  as  almost  invariably  happens  when  the  fresh  virus  is 
used,  and  they  disappeared  in  a  shorter  time.  Thus  it  appears 
that  while  the  virus  may  have  been  alive  in  the  culture  media, 
it  had  evidently  become  more  or  less  attenuated.  Other 
attempts  to  infect  monkeys  with  cultures  have  failed,  as  also 
may  be  seen  from  Table  ii.  Experiments  in  cultivation  were 
continued,  but  with  no  further  success.  The  virus  has  be- 
come increasingly  more  and  more  attenuated  in  its  successive 
passage  through  monkeys,  and  it  has  become  increasingly 
difficult  to  obtain  sufficient  satisfactory  material  from  which 
to  prepare  the  cultures.  The  most  satisfactory  material  for 
work  in  cultivation  of  the  virus  can  only  be  secured  directly 
from  the  human  cases.  The  cultivation  of  the  virus  of  verruga, 
then,  seems  to  be  today  in  a  somewhat  similar  position  to  that 
of  the  virus  of  small-pox. 

Filterable  Qualities  of  the  Virus 

The  experiments  performed  in  relation  to  demonstrating  the 
fllterable  qualities  of  the  virus  obtained  from  human  lesions 
have  been  described  previously.     From  this  description,  and 


FILTERABLE  QUALITIES   OF   VIRUS  127 

from  Table  i,  it  may  also  be  seen  that  those  rabbits  inoculated 
with  the  diluted  filtrates  of  suspensions  made  by  grinding  the 
human  lesions  in  saline  solution  developed  no  lesions.  These 
experiments  are  not  conclusive,  however,  since  as  has  been  em- 
phasized only  a  portion  of  the  rabbits  inoculated  with  the  virus 
itself  by  the  intratesticular  method  develop  lesions.  This 
fact  is  also  shown  in  Table  i,  and  the  reason  for  this  result 
has  alread}^  been  considered  in  this  report.  No  monkeys  were 
available  for  filtration  experiments  while  human  material  was 
available.  Although  we  had  been  informed  that  monkeys 
were  plentiful  in  Peru,  we  had  the  greatest  difficulty  in  obtain- 
ing them  upon  the  western  coast  of  South  America,  and  it  was 
necessary  to  import  them  via  Panama.  Subsequently,  how- 
ever, experiments  were  performed  relating  to  the  filterable 
qualities  of  the  virus  with  material  obtained  from  the  lesions 
produced  in  monkeys  by  inoculation  of  the  virus  after  numer- 
ous successive  passages  of  it  through  these  animals.  In  the 
experiments  in  which  portions  of  the  verruga  nodules  from 
monkeys  were  finely  ground,  suspended  in  water  or  saline 
solution,  and  the  resulting  mixture  passed  through  Berkefeld 
filters  (No.  2)  the  filtrates  failed  to  produce  lesions  in  monkeys, 
and  monkeys  inoculated  with  such  filtrates  were  not  rendered 
immune  to  subsequent  infection  with  the  unfiltered  virus. 
These  experiments  are  also  summarized  in  Table  ii.  Either 
the  virus  was  present  in  too  small  amount  in  the  filtrates,  or 
was  in  too  attenuated  a  condition  to  give  rise  to  the  production 
of  lesions  in  an  animal  so  relatively  insusceptible  to  the  infec- 
tion as  is  the  monkey,  or  else  it  had  failed  to  pass  through  the 
pores  of  the  filter.  Nevertheless,  we  have  been  unable  to 
detect,  as  has  been  shown  in  many  of  the  experiments  recorded, 
by  microscopic  examination  either  in  numerous  fresh  prepara- 
tions, often  examined  with  the  dark-field  microscope,  or  in 
stained  preparations  made  from  the  verruga  lesions,  or  in 
cultures  made  from  them,  or  in  sections  from  the  tissues,  any 
definite  visible  microorganism.  Tt  seems  not  unlikely  that 
the  virus  of  verruga  will  ultimately  be  shown  to  be  a  filterable 
one. 


128  VERRUGA  PERUVIANA 

As  is  well  known,  the  success  in  filtering  a  virus  or  organism 
depends  not  only  upon  the  form  and  dimension  of  it,  and  the 
texture  and  thickness  of  the  filter  used,  but  upon  the  pressure 
employed,  the  duration  of  the  filtration,  and  the  composition 
of  the  liquid  in  which  the  virus  or  organism  is  suspended. 
The  results  obtained  in  animals  inoculated  with  the  filtrates  of 
some  of  the  ''  filterable  viruses  "  are  frequently  not  constant, 
and  before  it  can  be  definitely  asserted  that  a  virus  is  non- 
filterable,  very  extensive  experimentation  with  a  large  amount 
of  material  is  sometimes  necessary. 

For  many  years  the  virus  of  small-pox  was  regarded  as  non- 
filterable,  and  in  many  experiments  made  in  connection  with 
the  filtration  of  this  virus,  the  filtrates  of  it  often  fail  to  produce 
lesions  in  monkeys.  Sims  Woodhead  ^  has  recently  again 
called  attention  to  this  fact  and  has  mentioned  that  during  an 
epidemic  of  small-pox  he  attempted  to  obtain  vaccine  by 
filtration  but  found  that  not  a  trace  of  active  vaccine  was 
present  in  the  filtrate  after  the  process  was  completed  whether 
the  dilution  was  high  or  low.  Further  experiments  in  relation 
to  the  question  of  the  filterability  of  the  virus  of  verruga 
shou^ld  be  performed  with  perfectly  fresh  material  obtained 
irout.  the  human  lesions,  and  the  inoculation  of  the  filtrates 
should  be  made  into  human  beings  or  into  monkeys  to  demon- 
strate whether  the  virus  has  passed  through  the  pores  of  the 
filter  in  sufficient  amount  to  produce  lesions.  Mayer,  Rocha- 
Lima  and  W^erner  ^  have  performed  a  single  experiment 
regarding  the  filterability  of  the  verruga  virus.  In  this  in- 
stance the  filtrate  from  a  Berkefeld  candle  of  a  suspension  of 
four  ground  papules  removed  from  their  case  was  inoculated 
over  the  eyes  of  a  monkey.  The  experiment  resulted  nega- 
tively, and  no  lesion  appeared.  We  feel  that  the  results  of 
the  experiments  regarding  the  non-filterable  qualities  of  the 
virus  after  its  repeated  passage  through  animals  which  have 
been  performed  upon  monkeys  should  not  be  considered  as 
conclusive.     In  connection  with  the  consideration  of  the  filter- 

1  Woodhead:  Proc.  xviith  Internat.  Cong.  Med.,  Lond.,  1913,  Sect.  4,  Bacteriology 
and  Immunity,  122. 

^  Mayer,  Rocha-Lima,  Werner:  loc.  cit. 


RELATION  TO  SMALL-POX  129 

able  qualities  of  the  virus  of  verruga  peruviana  the  important 
researches  of  Rous  '  relating  to  the  nature  of  the  filterable 
agent  causing  a  sarcoma  of  the  fowl  are  of  great  interest. 

Similarity  between  the  Virus  of  Verruga  and  of  Small-pox 

We  have  called  attention  to  the  fact  that  when  monkeys 
are  inoculated  with  the  virus  of  verruga  or  that  of  small-pox, 
that  only  a  modified  form  of  the  original  disease  results  and  in 
the  case  of  verruga,  only  a  locahzed  lesion  develops.     Neither 
the  virus  of  small-pox  nor  that  of  verruga,  as  we  have  shown, 
has  been  satisfactorily  cultivated  in  vitro  upon  artificial  media. 
In  several  other  ways  the  virus  of  verruga  shows  some  simi- 
larity to  that  of  small-pox,  but  in  other  ways  obviously  the 
two  behave  very  differently.     So  far  no  definite  visible  micro- 
organism has  been  discovered  in  veriuga  peruviana,  although 
Alayer,  Rocha-Lima  and  Werner  ^  in  the  study  of  a  single 
case,  reported  the  presence  of  bodies  resembling  chlamydozoa 
in  the  lesions.     The  cell  inclusions  observed  in  variola  and 
vaccinia  have  also  been  referred  by  Prowazek  ^  to  the  chlamy- 
dozoa.    Schilling-Torgau  4    places    both    verruga    peruviana 
and  variola  in  the  list  of  diseases  caused  by  filterable  viruses, 
but  he  gives  no  evidence  to  support  the  view  that  the  virus 
of  verruga  is  filterable.     Apart  from  the  evidence  obtained 
from  our  own  experiments  and  the  single  negative  one  per- 
formed by  Mayer,  Rocha-Lima,  and  Werner,  already  referred 
to   on  page   128,   no   experiments  relating  to   this  question 
apparently  have  been  performed.     The  filterable  properties 
of  the  virus  of  verruga  have,  however,  already  been  discussed 
upon  page  126.     The  virus  of  verruga  is  much  less  stable  than 
that  of  small-pox  and  after  the  verruga  virus  from  animals 
has  been  glycerinized  even  for  a  short  time  it  is  at  least  some- 
times no  longer  inoculable  for  monkeys,  as  we  have  shown 

'  Rous:  J.  Am.  M.  Ass.,  Chicago,  1911,  Ivi,  198;  1912,  Iviii,  19.38. 

"  Mayer,  Rocha-Lima,  Werner:  Munchen.  med.  Wchnschr.,  1913,  No.  14;  Ver- 
hundl.  d.  deutsch.  path.  Gesell.sch.,  Miirz-April,  1913,  198. 

'  Prowazek:  Ilandljuch  der  PathoKcnen  Protozoen,  Leipz.,  1912,  Erster  Band,  119. 

*  SchillinK-TorKau:  Mense,  Handbuch  der  Tropenkrankheiten,  Leipz.,  1914  Zweite 
Auflage,  Zweiter  Band,  1 10. 


130  VERRUGA  PERUVIANA 

(see  Table  ii).  The  two  viruses  show  similarity,  however, 
in  the  fact  that  monkeys  which  have  been  successfully  inocu- 
lated with  either  of  them  are  subsequently  usually  immune 
to  a  second  inoculation  of  the  corresponding  virus.  The 
verruga  virus,  on  the  other  hand  produces  no  lesion  when 
inoculated  upon  the  rabbit's  cornea,  as  does  the  variola  one. 
The  disease  produced  in  man  by  the  small-pox  virus  is  of  a 
more  virulent  type  than  that  produced  by  the  one  of  verruga. 
The  clinical  appearance  and  structure  of  the  lesions  upon 
the  skin  are  also  entirely  different  and  small-pox  is  very 
contagious.  The  inoculation  of  man  with  small-pox  virus 
obtained  directly  from  a  human  case  of  small-pox,  however, 
is  not  usually  a  dangerous  proceeding,  and  we  have  shown  in 
one  case  that  the  inoculation  of  a  man  with  verruga  virus 
obtained  directly  from  another  human  case  was  accomplished 
without  serious  result.  After  considering  these  facts  it  seems 
apparent  that  there  is  a  slight  analogy  between  the  two  viruses. 
However,  the  unique  character  of  verruga  peruviana  perhaps 
impresses  one  as  more  remarkable  than  its  similarity  to  any 
other  disease.  Reference  has  already  been  made  to  certain 
tumors  of  the  fowl  described  by  Rous  as  due  to  a  filterable 
virus.     (Page  129.) 

Differential  Diagnosis  of  Verruga  Peruviana 

Differentiation  from  Framboesia. —  It  has  been  suggested 
that  verruga  peruviana  is  but  a  form  of  framboesia.  Thus 
Eder  ^  inclines  to  the  opinion  that  the  two  diseases,  yaws  and 
verruga,  are  nearly  related,  verruga  being  nothing  more  than 
a  severe  form  of  framboesia  or  yaws,  modified  partly  by  the 
high  altitude  of  the  regions  where  it  occurs,  and  partly  by  being 
complicated  with  malaria.  Biffi,^  however,  who  has  observed 
verruga  peruviana  in  Peru  is  entirely  opposed  to  this  view. 
In  our  opinion,  any  one  who  has  had  a  wide  experience  with 
either  disease  would  not  confuse,  from  a  clinical  standpoint, 
the  one  with  the  other.     Only  in  the  distribution  of  the  lesions 

1  Eder:  J.  Trop.  Med.,  Lond.,  1906,  ix,  213. 

2  Biffi:  Arch.  Schiffs-  u.  Tropenhyg.,  Leipz.,  1908,  xii,  1. 


DIFFERENTIAL   DIAGNOSIS  131 

sometimes,  and  in  the  fact  that  in  both  small  nodular  tumors 
appear  upon  the  skin,  is  there  any  resemblance  in  the  two 
diseases.  From  the  descriptions  of  the  lesions  already  given 
on  pages  65  to  81  this  fact  should  appear  evident.  The 
most  characteristic  feature  of  the  yaws  lesion  is  that  the 
primary  one  is  a  papule  which  after  about  a  week  becomes 
moist,  developing  a  yellowish  secretion  which  dries  into  a 
crust.  The  subsequent  lesions  are  also  commonly  crusted. 
The  verruga  has  an  entirely  different  appearance.  It  is 
practically  never  crusted,  as  we  have  seen  from  the  descrip- 
tions already  given.  Indeed,  the  most  characteristic  feature 
in  its  appearance  is  that  the  skin  is  smooth,  tense,  and  trans- 
lucent over  it,  and  that  the  external  surface  of  the  lesion  at 
the  height  of  the  disease  resembles  very  much  a  cherry  on 
account  of  its  color  and  translucence. 

Other  points  of  differentiation  between  the  two  diseases 
which  have  been  referred  to  are  that  framboesia  is  much  more 
contagious  than  verruga.  In  framboesia  there  is  generally 
a  distinguishable  point  where  the  initial  lesion  developed.  In 
verruga  this  is  not  so  apparent.  However,  as  w^e  have  shown 
elsewhere,  there  are  other  more  decisive  points  of  difference. 
Thus  from  an  etiological  and  pathological  standpoint  the  two 
are  entirely  distinct  infections.  In  a  very  careful  study  of  the 
lesions  of  verruga  peruviana,  as  we  have  emphasized  in  the 
experiments  discussed  on  pages  82-122,  we  have  been  unable  to 
detect  Spirochaeta  pertenuis  or  any  other  visible  microorgan- 
ism. In  the  very  numerous  experiments  in  which  fresh  mate- 
rial from  the  human  verruga  lesions  has  been  injected  into 
the  rabbit's  testis,  the  lesions  produced  have  not  resembled 
those  which  result  from  the  inoculation  of  material  from  the 
lesions  of  framboesia  cases,  and  again  no  spirochaetae  have 
been  found  in  them.  In  the  intratesticular  lesion  which  results 
trom  the  inoculation  of  framboesia  material,  as  was  first  shown 
by  Nichols,^  spirochaetae  are  abundant.  Finally,  the  disease 
verruga  peruviana  is  not  cured  by  injections  of  salvarsan, 
as  was  first  shown  by  one  of  us   (Strong)  -  in   1910,  to  be 

'  Nichols:  J.  Exper.  Med.,  1910,  xii,  616. 

2  Strong:   Philippine  J.  Science,  Manila,  1910,  v,  433. 


132  VERRUGA   PERUVIANA 

invariably  the  case  with  framboesia.  The  lesions  of  the 
skin  of  verruga  peruviana  seem  to  be  entirely  uninfluenced 
by  injections  of  this  substance,  while  in  framboesia  they 
disappear  very  rapidly  following  its  injection. 

It  is  not  deemed  necessary  here,  to  consider  in  detail  the 
differentiation  between  the  histological  structure  of  the  lesions 
of  verruga  and  those  of  framboesia.  In  framboesia  the  lesions 
affect  the  epithelium  rather  than  the  cutis.  The  surface 
epithelium  is  greatly  increased  and  the  epithelial  cells  are 
degenerated  and  swollen.  In  verruga  the  most  striking  fea- 
ture is  the  extensive  endothelial  proliferation  occurring  about 
newly  formed  blood-vessels,  a  condition  which  is  not  observed 
in  framboesia.  The  histological  appearances  of  the  verruga 
lesions  are  thoroughly  considered  on  page  145,  and  from 
this  description  it  is  evident  that  from  a  pathological  stand- 
point, as  well  as  from  a  clinical  one,  the  two  diseases  are  also 
distinguishable. 

While  discussing  the  differentiation  of  verruga  peruviana 
from  framboesia,  it  may  be  advisable  briefly  to  refer  to  its 
differentiation  from  syphilis.  From  what  has  already  been 
said  regarding  the  absence  of  spirochaetae  in  not  only  the 
human  lesions  of  verruga,  but  particularly  in  those  produced 
in  the  rabbit's  testis  with  verruga  material,  and  from  the 
complete  resistance  of  the  disease  to  treatment  with  salvarsan, 
it  would  appear  evident  that  the  disease  is  not  a  form  of 
syphilis.  The  Wassermann  reaction  was  negative  with  one 
exception  in  the  cases  of  verruga  which  we  studied.  In  per- 
forming these  experiments  cases  of  syphilis  were  used  as  posi- 
tive controls,  and  normal  cases  for  negative  controls.  In 
nine  cases  of  verruga  in  which  a  well-marked  eruption  was 
present,  the  reaction  of  the  serum  was  also  investigated  in 
relation  to  the  fixation  of  complement  with  a  specific  antigen 
prepared  from  a  verruga  nodule.  In  none  of  these  cases  was  a 
fixation  of  complement  obtained.  It  is  noteworthy  in  this 
connection    that    according    to    Castellani  ^    and    Bowman  ^ 

1  Castellani:  J.  Hyg.,  Camb.,  1907,  vii,  566. 

2  Bowman:  Philippine  J.  Science,  Manila,  1910,  v,  485. 


DIFFERENTIAL  DIAGNOSIS  133 

fixation  of  complement  occurs  with  the  serum  of  yaws  cases 
when  yaws  antigen  is  employed.  Baermann  ^  found  that  in 
yaws  as  in  sj'philis  the  Wassermann  reaction  is  present. 

In  the  study  of  the  blood  in  verruga  peruviana,  it  was  found 
that  an  extract  prepared  from  a  cutaneous  nodule  from  a  case  of 
verruga  showed  the  presence  of  a  hemolysin  which  was  active 
in  dilutions  of  1  to  1,000. 

Differentiation  from  Oroya  Fever.  —  From  the  descriptions 
given  in  Sections  III  and  IV,  regarding  Oroya  fever  and 
verruga  peruviana,  and  from  the  experiments  detailed  in  these 
sections,  it  would  seem  evident  that  Oroya  fever  and  verruga 
peruviana  are  two  distinct  diseases.  We  believe  that  we  have 
conclusively  shown  in  these  pages,  first,  that  verruga  peruviana 
is  due  to  a  virus  which  may  be  transmitted  to  several  of  the 
lower  animals,  particularly  monkeys,  in  which  animals  charac- 
teristic lesions  are  developed;  -  second,  that  Oroya  fever  is  due 
to  a  microorganism  {Bartonella  bacilli formis)  which  is  a 
parasite  of  the  red  blood-corpuscles  and  of  the  endothelial 
cells.  Up  to  the  present  time  it  has  not  been  transmitted  to 
animals.  In  man  in  severe  infections  this  parasite  gives  rise 
to  a  rapid  and  intense  anaemia  which  often  results  fatally. 
Verruga  peruviana  is  very  rarely  a  fatal  disease,  and  there  is 
no  such  marked  anaemia  accompanying  it.  Its  most  character- 
istic feature  is  the  occurrence  of  the  eruption  upon  the  skin. 
When  fresh  material  from  the  lesions  of  verruga  is  inoculated 
intravenously  and  cutaneously  into  monkeys,  the  parasite  of 
Oroya  fever  does  not  appear  in  the  blood  of  these  animals. 
The  pathology  of  the  two  diseases  is  also  quite  distinct,  as 
is  evident  from  the  facts  set  forth  regarding  this  question  in 
Section  III,  page  42  and  Section  IV,  page  137.  Ba?-- 
tonella  bacilliformis  is  not  found  in  the  blood  of  uncompli- 
cated cases  of  verruga  peruviana,  but  occasionally  concomitant 
infections  of  Oroya  fever  and  of  verruga  are  encountered  just 
as  concomitant  infections  with  verruga  and  malaria  are 
frequently  observed.     This  fact  has  been  more  fully  discussed 

'  Baermann:  Miinchen.  med.  Wchnschr.,  1910,  No.  41. 

2  The  characteristics  of  this  virus  have  been  described  on  pages  122  to  130. 


134  VERRUGA  PERUVIANA 

on  page  64.  In  spite  of  the  evidence  which  we  had  adduced 
of  the  specificity  of  the  two  diseases,  many  Peruvian  physi- 
cians insisted  upon  their  unity  and  claimed  that  in  Carrion's 
experiment  the  inoculation  was  made  with  blood  from  a 
verruga  nodule,  and  that  he  died  of  Oroya  fever.  On  account 
of  the  insufficient  data  regarding  this  experiment,  the  condi- 
tion present  at  the  autopsy,  and  the  examination  of  the  blood, 
it  is  impossible  to  say  today  of  what  infection  Carrion  died. 
Certainly  there  is  no  definite  proof  that  he  died  of  Oroya  fever. 
Nevertheless,  it  seemed  advisable  to  repeat  the  experiment  of 
Carrion.  Having  become  convinced  from  clinical  observation 
and  experimental  evidence  that  Oroya  fever  and  verruga 
peruviana  were  two  entirely  distinct  diseases,  and  having 
secured  through  the  kind  assistance  of  Dr.  Matto,  Professor 
of  Bacteriology  in  the  University  of  Lima,  a  volunteer  (a 
Chilean),  a  direct  inoculation  was  made  from  two  cases  of 
verruga  peruviana.  A  portion  of  a  skin  lesion  from  each  of 
the  two  patients  with  verruga  was  removed,  and  within 
twenty  minutes  of  the  time  of  this  operation  the  skin  of  the 
normal  person  over  the  left  shoulder  was  thoroughly  scarified, 
and  a  portion  of  the  nodules  removed  from  the  verruga 
patients  thoroughly  rubbed  into  the  scratches.  The  vaccina- 
tion scratches  were  healed  entirely  at  the  end  of  about  ten 
days,  and  the  skin  appeared  normal.  On  the  sixteenth  day 
two  small  groups  of  cherry-red  papules  at  the  end  of  some  of 
the  vaccination  scratches  appeared,  and  a  few  days  later 
another  small  group  became  visible.  At  this  time  the  note 
made  regarding  the  experiment  states: 

"  Today  the  papules  measure  from  1  to  2  mm.  in  diameter,  they  are  situated  in 
three  clusters,  at  the  ends  of  the  vaccination  scratches.  The  scratches  have  all 
healed  and  the  skin  appears  otherwise  normal.  The  papules  are  of  a  bright  red 
color,  raised  above  the  surface  of  the  skin,  and  sharply  circumscribed.  The  skin 
covering  them  is  tense  and  translucent." 

These  papules  increased  slowly  in  size  until  the  thirty-fifth 
day,  when  two  of  the  groups  were  removed.  The  blood 
examination  of  the  vaccinated  person  never  revealed  the 
parasite  observed  in  the  blood  of  Oroya  fever  cases,  and  no 
appreciable    anaemia    developed.     The  red  blood-corpuscles 


DIFFERENTIAL   DIAGNOSIS  135 

numbered  4,300,000  per  cubic  mm.  at  the  time  of  the  inocula- 
tion, and  at  the  time  of  the  appearance  of  the  eruption  they 
were  4,500,000.  On  the  day  the  papules  were  removed  the 
red  count  was  4,216,000.  The  leukocytes  numbered  10,000 
on  the  day  the  papules  were  first  observed.  After  a  week  the 
count  showed  8,000.  There  was  but  a  slight  febrile  reaction 
preceding  the  papular  eruption  and  the  symptoms  preceding 
it  were  mild.  The  patient  stated  that  he  felt  languid,  and  the 
facial  expression  gave  the  appearance  of  his  being  tired  and 
depressed.  No  generalized  eruption  upon  the  skin  occurred, 
and  the  individual  was  in  good  health  at  the  time  of  our 
departure  from  Lima.  A  portion  of  the  lesions  removed  from 
the  case  was  placed  in  Zenker's  solution  for  section,  and 
another  portion  ground  and  suspended  in  saline  solution, 
and  inoculated  into  the  testis  of  Rabbit  No.  73.  The  sections 
of  the  lesions  showed  distinct  papular  formation  and  a  similar 
histological  structure  to  that  observed  in  other  early  human 
lesions  of  the  disease.  The  rabbit  developed  a  typical  verruga 
lesion  in  the  testis  after  twenty-five  days.  From  this  experi- 
ment it  appears  evident  that  the  direct  inoculation  of  the 
verruga  virus  from  man  to  man  does  not  produce  Oroya  fever, 
but  a  modified  form  of  verruga  eruption,  just  as  the  inocula- 
tion of  small-pox  virus  from  man  to  man  produces  a  modified 
form  of  small-pox  eruption.  From  this  experiment  it  would 
also  appear  that  the  last  objection  to  the  idea  that  verruga 
peruviana  and  Oroya  fever  are  two  distinct  diseases  as  based 
upon  Carrion's  experiment  has  been  removed. 

Relationship  to  Paratyphoid  Fever. —  It  has  been  suggested 
that  Oroya  fever  is  paratyphoid  to  which  yaws  is  superadded. 
This  statement,  it  appears  to  us,  requires  no  further  discussion 
here.  We  have  already  referred  to  the  fact  on  page  39  that 
paratyphoid  fever  has  often  been  diagnosed  in  Peru  under  the 
name  of  Carrion's  fever,  and  sometimes  as  Oroya  fever.  Of 
the  twenty-four  cases  of  verruga  peruviana  which  we  studied, 
none  of  them  were  complicated  with  paratyphoid  infection. 
Of  course  there  is  always  a  possibility  that  a  concomitant 
infection  with  these  two  diseases  might  occur.     During  the 


136  VERRUGA  PERUVIANA 

time  our  investigations  in  Lima  were  being  made,  we  observed 
a  case  of  fever  without  any  eruption  which  was  shown  to  us 
as  Oroya  fever.  We  found  no  microorganisms  in  the  red 
blood-corpuscles  of  this  patient.  Subsequently  we  were  able 
to  show  by  bacteriological  examination  that  the  case  was  one 
of  paratyphoid  infection,  and  not  one  of  Oroya  fever. 

From  the  discussion  given  in  this  section  of  the  report  it 
seems  evident  that  verruga  peruviana  is  a  distinct  disease  sui 
generis. 

Relationship  to  Angiofibroma  Cutis  Contagiosum.  —  Basse- 
witz  ^  has  described  in  Brazil  under  the  name  of  "  angiofibroma 
cutis  circumscriptum  contagiosum  "  a  disease  which  from  the 
description  given  evidently  bears  considerable  resemblance  to 
verruga  peruviana.  Bassewitz  states  that  from  the  appearance 
of  the  lesions  upon  the  skin  alone  a  differential  diagnosis  of  the 
two  is  difficult.  No  spirochaetae  were  found  in  the  lesions.  He 
believes,  however,  that  this  angiofibroma  is  a  different  disease 
from  verruga,  basing  his  views  upon  the  descriptions  given  in 
the  literature  regarding  the  symptoms  of  verruga  peruviana. 
He  calls  attention  to  the  fact  that  there  is  not  the  same  high 
mortality  in  angiofibroma  contagiosum,  that  the  eruption  is 
not  preceded  by  high  fever,  anaemia,  and  prostration,  and  that 
symptoms  due  to  the  development  of  the  lesions  internally  also 
do  not  occur.  Rocha-Lima  ^  in  a  more  recent  publication  gives 
these  same  views  as  his  own  although  he  has  never  apparently 
had  an  opportunity  of  examining  the  fresh  blood  in  a  case  of 
Oroya  fever  and  has  studied  but  one  case  of  verruga  peruviana. 
We  have  pointed  out  above  that  these  symptoms  do  not 
occur  in  uncomplicated  verruga  peruviana,  and  the  mortality 
of  the  disease  is  not  high.  From  a  histological  as  well  as  a 
clinical  standpoint  it  appears  that  the  two  conditions  if  not 
identical  are  probably  very  closely  related.  The  histology 
of  both  is  considered  on  page  151.  Further  information, 
however,  is  desirable  before  verruga  peruviana  and  angio- 
fibroma circumscriptum  contagiosum  can  definitely  be  re- 
garded as  one  and  the  same  disease. 

1  Bassewitz:  Arch.  f.  Schiffs-  u.  Tropenhyg.,  Leipz.,  1906,  x,  201. 

2  Rocha-Lima:  loc.  cit. 


VERRUGA   PERUVIANA  137 

Pathology 

The  distribution  and  description  of  the  lesions  as  they  occur 
upon  the  skin  and  visible  mucous  membranes  have  already 
been  referred  to  at  some  length  on  pages  65  to  81.  When 
the  smaller  papular  lesions  are  removed,  the  tumors  are  seen 
to  be  rounded  or  oval.  The  cut  surface  is  very  moist,  and 
serum  and  a  variable  amount  of  blood  escape  as  the  section  is 
being  made.  In  the  more  acute  lesions  the  cut  surface  of  the 
superior  portion  of  the  papule  usually  is  of  a  bright  cherry-red 
color,  while  at  the  base  the  tissue  has  a  grayer  appearance. 
Under  a  hand  lens  the  cut  surface  of  the  papule  appears  moist, 
smooth,  homogeneous,  and  very  translucent.  No  blood- 
vessels of  sufficient  size  to  be  recognized  with  a  hand  lens  are 
present,  and  no  lobular  appearance  is  observable. 

Some  of  the  larger  lesions  measuring  from  1.5  to  2  cm.  in 
diameter,  after  removal  present  a  different  appearance.  These 
may  be  rounded  or  dome-shaped.  In  the  so-called  mulaire 
lesions  the  skin  covering  the  nodule  sometimes  resembles 
somewhat  an  onion  skin.  This  appearance  is  given  by  the 
fact  that  the  superficial  layers  of  the  skin  have  become  slightly 
dry  and  the  surface  has  become  traversed  by  multiple 
longitudinal  lines.  These  lesions  after  removal  usually  present 
a  variegated  appearance,  being  bluish,  grayish,  or  purplish  in 
color.  On  cut  section  through  the  middle  of  the  larger  tumors 
the  surface  is  also  moist,  and  serum  and  blood  exudes.  The 
cut  surface  in  general  is  dark  cherry-red,  traversed  by  longi- 
tudinal gray  lines.  Sometimes  yellowish-gray  areas  are  seen 
in  the  substance  with  gray  lines  radiating  about  them.  Under 
the  microscope  newly  formed  connective  tissue  is  seen  to 
occupy  the  areas  in  which  the  gray  lines  are  present,  and 
evidently  gives  rise  to  the  appearance  described.  The  yellow- 
ish-gray areas  are  seen  microscopically  to  comprise  the  more 
compact  cellular  portions  of  the  nodule.  A  few  very  small 
blood-vessels  can  be  distinguished  with  the  naked  eye  in  the 
larger  lesions.  Sometimes  the  skin  has  become  quite  adherent 
over  the  tumor,  and  in  the  cut  section  in  these  instances  some 


138  VERRUGA  PERUVIANA 

of  the  gray  lines  radiating  from  the  surface  evidently  represent 
an  invasion  of  epithelium  into  the  depth  of  the  tumor  for  a 
distance  of  several  millimeters.  The  larger  mulaire  lesions 
sometimes  show  superficial  ulceration  and  present  then  a 
reddened,  moist  surface.  The  appearance  of  these  lesions  is 
obviously  somewhat  niodified  by  secondary  infections  with 
bacteria.     Deep  ulceration  practically  never  occurs. 

The  distribution  of  the  lesions  upon  the  skin  and  visible 
mucous  membranes,  the  lips,  gums,  conjunctivae  and  glans 
penis  has  already  been  described. 

In  the  description  of  the  disease  given  in  the  textbooks,  one 
usually  finds  the  statement  that  the  eruption  occurs  in  the 
internal  organs.     Thus  Scheube  ^  writes, 

"  There  exists  no  mucous  membrane  or  organ  in  which  they  may  not  appear, 
and  the  symptoms  they  can  originate  are  remarkably  manifold.  Thus  excrescences 
in  the  nose  may  cause  epistaxis;  in  the  throat  and  oesophagus,  dysphagia;  if 
situated  in  the  larynx,  cough,  hoarseness,  haemoptysis,  and  even  suffocation  may 
result;  if  they  have  their  seat  in  the  lungs  haemoptysis  and  infiltration  may  set  in, 
■which  may  be  mistaken  for  tuberculosis;  when  situated  in  the  stomach  they  cause 
haematemesis;  in  the  intestine,  diarrhoea  and  intestinal  haemorrhage;  in  the 
kidneys  or  bladder,  haematuria;  and  in  the  uterus,  metrorrhagia.  In  rare  cases 
the  brain  and  spinal  column  participate  in  the  disease,  inducing  epileptiform  con- 
vulsions and  symptoms  of  meningitis." 

The  idea  of  the  occurrence  of  the  eruption  in  the  internal 
organs  appears  to  have  been  passed  on  in  the  literature  from 
author  to  author  since  1845,  when  Tschudi  ^  in  addition 
reported  the  occurrence  of  the  lesions  in  the  bones.  In  our 
opinion  the  evidence  given  regarding  the  occurrence  of  the 
lesions  of  verruga  peruviana  in  the  internal  organs  is  not  con- 
clusive. The  anatomical  changes  that  have  been  found  in 
the  solid  viscera  in  this  disease  appear  frequently,  at  least, 
to  have  resulted  from  complication  with  other  diseases.  Thus 
it  is  evident  that  the  internal  lesions  of  tuberculosis  have 
sometimes  been  mistaken  for  those  of  verruga  peruviana. 
The  tissues  of  the  organs  from  Cases  B  and  X,  reported  upon 
on  page  54,  in  which  the  diagnosis  of  Oroya  fever  and  verruga 
peruviana  had  been  made  during  life  by  the  physicians  who 
observed  them,    evidently   came   from   individuals   suffering 

1  Scheube:   loc.  cit.,  302.  2  Tschudi:   loc.  cit. 


PATHOLOGY  139 

with  tuberculosis.  This  fact  we  were  able  subsequently  to 
show  from  a  microscopical  study  of  the  sections  of  these 
organs.  We  were  also  able  to  demonstrate  acid-fast  bacilli 
in  these  sections.  During  our  stay  in  Peru  in  several  in- 
stances, miliary  tubercles  in  the  spleen  and  liver,  due  to  tuber- 
culosis, were  shown  us  by  phj^sicians  as  examples  of  the  internal 
lesions  of  verruga  peruviana.  Attention  already  has  been 
called  to  the  fact  that  NicoUe,  Letulle,  and  Galli  Valerio 
found  acid-fast  bacilli  in  the  lesions  of  verruga,  and  that 
Darling  observed  at  autopsy  a  case  of  verruga  with  generalized 
tuberculosis  with  lesions  in  the  lungs,  pleurae,  lymph-nodes, 
spleen,  kidneys,  and  liver.  Nicolle  ^  in  a  case  diagnosed  as 
maladie  de  Carrion  with  internal  verruga  lesions  found  acid- 
fast  bacilli  resembling  the  tubercle  bacillus  in  lesions  in  the 
liver,  lungs,  spleen,  and  lymphatics.  Giant-cells  were  observed 
in  manj^  of  the  tubercles.  Bindo  De  Vecchi  -  studied  micro- 
scopically the  tissues  from  two  cases  of  verruga  peruviana 
said  to  have  shown  at  autopsy  internal  lesions.  The  tissues 
were  sent  to  him  from  Peru.  In  the  first  case  which  had  high 
fever  during  life  a  lymphatic  abscess  was  said  to  have  been 
present  from  which  a  paracolon  bacillus  was  isolated.  Re- 
garding the  second  case  no  definite  clinical  information  was 
furnished.  In  both  of  these  cases  in  addition  to  the  skin  lesions 
De  Vecchi  found  lesions  in  the  liver,  spleen,  and  lungs.  These 
internal  lesions  consisted  of  nodules  showing  necrosis  with  for- 
mation of  fibroblasts  and  giant-cells.  The  pictures  of  the  inter- 
nal lesions  in  the  liver  and  spleen  which  De  Vecchi  publishes 
are  very  different  from  the  verruga  lesions  as  we  have  observed 
them.  He  also  observed  bacilli  present  in  many  of  these 
lesions.  During  our  stay  in  Lima,  through  the  kindness  of 
several  Peruvian  physicians,  and  particularly  through  that 
of  Dr.  Hercelles,  we  had  opportunity  of  examining  all  of  the 
pathological  material  present  in  the  museum  of  the  Dos  de 
Mayo  Hospital  relating  to  verruga  peruviana  and  Oroj^a 
fever.     None  of  the  lesions  we  observed  in  the  internal  organs 

»  Nicolle:  loc.  cit.,  593. 

*  De  Vecchi:  Beiheft  4,  Arch.  f.  Schiffs-  u.  Tropenhyg.,  Leipz.,  1909,  xiii,  143. 
Virchow's  Arch.  f.  path.  Anat.,  Berl.,  1908,  c.Kciv,  Beiheft,  1. 


140  VERRUGA  PERUVIANA 

were  convincing  that  they  were  of  a  verruga  nature.^  Only 
in  two  of  the  cases  did  it  seem  that  there  was  any  question  as  to 
whether  the  lesions  were  those  of  verruga  peruviana.  In  one  of 
these  a  number  of  small  tumors,  measuring  about  4  mm.  in  di- 
ameter,were  situated  in  the  heart  beneath  the  pericardium,  and 
in  another  specimen  from  another  case  there  were  small  miliary 
nodules  in  the  pharynx  and  larynx.  A  description  of  the 
histological  examination  of  the  sections  from  these  lesions  has 
been  given  on  page  53.  Microscopical  examination  of  the  one 
from  the  heart  showed  multiple  cysts  caused  apparently  by  a 
cestodal  infection.  In  sections  of  the  other  specimen  from  the 
pharynx  numerous  rounded  masses  of  vascular  tissue  were  pres- 
ent. There  apparently  was  no  tendency  to  caseation  and  no 
giant-cells  were  discovered.  No  acid-fast  bacilli  were  found 
in  the  lesions.  It  seems  probable  that  these  lesions  in  the 
pharynx  and  larynx  represent  those  of  verruga  peruviana, 
and  that  the  eruption  of  the  disease  occurred  not  only  in  the 
mouth,  but  also  in  the  pharynx  and  upper  part  of  the  larynx. 
No  lesions  were  found  in  sections  of  the  spleen  from  this  case. 

Histology 

In  examining  the  literature  we  find  that  the  histology  of  the 
verruga  nodules  of  the  skin  has  been  studied  by  the  following 
investigators. 

Previous  Investigations.  —  Velez  ^  expressed  the  opinion  that 
the  cutaneous  verruga  nodules  originated  in  the  papillary 
layer  of  the  skin  and  mucous  membranes.  Dounon,  Cornil, 
and  Renaut  ^  from  the  study  of  sections  of  the  nodules  con- 
cluded that  they  were  of  a  fibrosarcomatous  structure,  being 
composed  largely  of  embryonic  connective  tissue. 

Izquierdo  ^  described  the  connective  tissue  structure  of 
some  of  the  tumors,  and  noted  their  resemblance  to  sarcoma. 

1  In  connection  with  this  statement  reference  may  be  made  to  the  review  of  Her- 
celles'  article  (Trop.  Dis.  Bull.,  Lond.,  1914,  Dec.  15,  iv,  486)  which  has  appeared 
while  this  report  was  in  press. 

2  Velez:  Gac.  med.  de  Lima,  1861,  v,  198. 

^  Dounon,  Cornil,  Renaut:  Arch,  de  med.  nav.,  Par.,  1871,  255. 
*  Izquierdo:  Virchow's  Arch.  f.  path.  Anat.,  Berl.,  1885,  xcix,  411. 


PATHOLOGY  141 

He  emphasized  the  vascularity  of  some  lesions  and  mentions 
a  true  cavernous  structure  in  some  portions  of  others.  He 
points  to  the  great  resemblance  of  these  to  the  true  cavernous 
angioma.  He  also  noted  the  presence  of  non  acid-fast  bacilli 
in  the  sections. 

LetuUe  ^  observed  subacute  proliferation  of  the  dermic  and 
subcutaneous  tissues,  together  with  hyperplasia  of  the  en- 
dothelial cells.  In  places  the  specific  elements  of  the  skin  had 
disappeared  entirely.  The  arteries  and  nerves  remained 
intact.  Other  areas  composed  of  lymphoid-cells  surrounded 
by  a  thin  endothelial  wall  were  also  noted.  Acid-fast  bacilli 
were  found  in  all  of  the  lesions. 

Nicolle  -  examined  histologically  fragments  of  liver,  kidney, 
lungs,  spleen,  and  lymphatics,  said  to  have  come  from  a  case 
of  the  Oroya  fever  type  in  which  there  were  only  internal 
verruga  lesions.  In  the  lungs  epithelioid  nodules  surrounded 
by  leukocytes  were  present.  There  was  no  caseation  and  no 
giant-cells  were  observed.  In  the  liver,  however,  giant-cells 
were  found,  but  no  nodules  and  no  necrosis.  The  lymphatic 
glands  showed  caseation,  and  similar  lesions  were  found  in 
the  spleen.  Acid-fast  organisms  resembling  the  tubercle 
bacillus,  from  a  morphological  standpoint,  were  found  in  these 
lesions. 

Hercelles  ^  attributed  a  vascular  origin  to  the  nodes,  believ- 
ing that  they  resulted  from  a  periarteritic  proliferation.  The 
regression  of  the  lesion  was  thought  to  be  due  to  an  endo- 
arteritic  process. 

Sometimes  giant-cells  were  observed  in  the  sections.  He 
believed  the  substance  of  the  verruga  nodules  to  be  formed 
from  a  reaction  which  resulted  in  the  massing  together  of  the 
tissue  cells. 

Escomel  ■*  believes  that  the  organism  of  verruga  is  carried 
in  the  blood  where  it  secretes  its  complex  toxin.     It  is  inter- 

1  Letulle:  M6m.  Soc.  de  hiol.,  Par.,  1898,  764. 

Odriozola's  MonoKraph,  201. 
'■  Nicolle:  Ann.  de  I'lnst.  Pasteur,  Par.,  1898,  xii,  590. 
'  Hercelles:  Th^se  de  Lima,  1900. 
*  Esconsel:  Ann.  de  dermat.  et  syph.,  Par.,  1902,  iii,  961. 


142  VERRUGA  PERUVIANA 

rupted  at  points  where  the  circulation  is  slow  and  where  there 
is  a  favorable  medium  such  as  the  skin  for  its  development. 
Leukocytes  accumulate  at  this  point,  and  a  proliferation  of 
the  connective-tissue  cells  occurs.  Later  an  increased  number 
of  newly  formed  blood-vessels  appears.  The  typical  verruga 
nodule  is  said  to  consist  of  verruga  cells,  "  cellules  verru- 
queuses  "  (formed  from  the  connective-tissue  cells),  few 
leukocytes,  and  a  connective  tissue  framework.  In  some 
lesions  many  red  blood-corpuscles,  leukocytes,  and  blood 
pigment  were  present.  In  the  regressive  ones  the  leukocytes 
were  not  abundant.  He  believes  the  verruga  nodule  belongs 
in  the  inflammatory  group  of  lesions  rather  than  among  the 
true  tumors.  Escomel  also  found  bacteria  present  in  many 
of  the  lesions  which  he  studied. 

De  Vecchi,^  as  has  been  mentioned  on  page  139,  studied 
tissues  sent  to  him  from  Peru  which  had  been  removed  from 
three  cases.  The  third  of  these  was  apparently  a  case  of  Oroya 
fever,  and  has  been  considered  on  page  52  of  this  report. 
No  verruga  nodules  were  found  in  the  internal  organs.  The 
first  case  was  complicated  with  pyogenic  infection.  There 
was  high  fever  during  life  and  swelling  of  the  lymphatic  glands. 
At  autopsy  the  spleen  was  greatly  enlarged,  and  in  the  sub- 
maxillary glands  an  abscess  containing  pus  was  present. 
There  were  also  nodules  in  the  skin  and  muscles,  and  tubercles 
in  the  lungs,  spleen  and  liver.  A  paracolon  bacillus  was  said 
to  have  been  isolated  from  the  pus  of  the  abscess.  Bacteria 
were  found  in  the  sections  of  the  internal  lesions  by  De  Vecchi. 
These  he  refers  to  as  bacilli  of  the  typhoid-colon  group.  In  one 
case  De  Vecchi  describes  the  lesions  in  the  spleen  as  multiple 
necroses.  Multinuclear  giant-cells  were  present  which  he 
states  resemble  the  giant-cells  seen  in  tuberculosis.  Some 
fibroblasts  were  present.  De  Vecchi  does  not  believe,  how- 
ever, that  the  lesions  were  produced  by  the  tubercle  bacillus. 
Similar  changes,  but  not  so  advanced,  were  found  in  the 
liver.      In   the    absence    of    animal   inoculations    the    non- 

1  De  Vecchi:  Beiheft  4,  Arch.  f.  Schiffs-  u.  Tropenhyg.,  Leipz.,  1909,  xiii,  143. 
Virchow's  Arch.  f.  path.  Anat.,  Berl.,  1908,  cxciv,  Beiheft,  1. 


PATHOLOGY  143 

tubercular  character  of  these  lesions  is  to  be  questioned.  In 
the  lungs  neither  degenerative  lesions  nor  giant-cells  were 
observed,  but  areas  were  present  in  the  center  of  which  the 
nuclei  stained  poorly.  The  reader  should  consult  De  Vecchi's 
article  for  the  full  description  of  these  changes.  Tubercle 
bacilli  were  not  found  in  the  lesions,  but  other  bacilli  single 
and  in  masses  were  present.  In  the  second  case  it  is  stated 
that  necrotic  changes  were  present  in  the  spleen,  though  of 
slight  degree.  The  nodules  in  the  liver  De  Vecchi  character- 
ized as  typical  of  verruga  lesions.  Lesions  were  also  found  in 
the  lungs  which  were  partly  necrotic,  and  a  bronchial  pneumo- 
nia was  present.  In  the  liver  and  spleen,  bacilli  were  found, 
and  in  the  lesions  of  the  lungs,  cocci  were  in  addition  present. 
Acid-fast  bacilli  were  not  found.  In  the  lesions  of  the  skin 
and  muscles  the  degenerative  changes  were  not  recognized, 
and  giant-cells  were  not  observed.  De  Vecchi  believes  that 
the  lesions  in  the  beginning  are  simply  of  a  hemorrhagic  nature, 
and  that  later  in  such  areas  there  is  a  proliferation  of  the 
connective  tissue  elements.  He  also  believes  that  the  necroses 
in  the  liver  and  spleen  result  from  other  hemorrhages  from 
the  newly  formed  blood-vessels.  The  most  striking  charac- 
teristic to  him,  regarding  the  verruga  lesions,  was  the  presence 
of  "  globulifere  "  cells, —  cells  formed  by  the  leukocytes  having 
engulfed  red  corpuscles.  He  also  refers  to  the  occurrence  of 
"  acidophile  "  bodies,  measuring  from  1  to  2  m  in  diameter 
which  he  believes  probably  originate  from  hemoglobin.  Blood- 
corpuscles  which  often  stained  poorly  were  observed  near 
these  areas.  The  verruga  nodule  he  regards  as  of  fibroblastic 
structure,  a  product  of  preexisting  elements  in  the  tissue  and 
the  expression  of  the  reaction  of  the  tissue  to  an  abnormal 
stimulus.  A  small  number  of  mono-  and  polymorphonuclear 
leukocytes  was  observed  in  the  lesions.  Newly  formed  blood- 
vessels were  almost  invariably  present  in  the  smaller  papules. 
More  recently  Rocha-Lima  ^  had  the  opportunity  of  studying 
histologically  lesions  from  the  skin  from  a  case  of  verruga 
peruviana  observed  in  Hamburg  with  Mayer  and  Werner. 

'  Rocha-Lima:   Verhandl.  d.  deutsch.  path.  Gesellsch.,  Jena,  1913,  198,  409. 


144  VERRUGA  PERUVIANA 

He  employed  modern  methods  of  technique,  and  his  descrip- 
tion of  the  histology  of  the  disease  in  our  opinion  is  the  most 
accurate  which  has  yet  been  given.  He  points  out  that  the 
compact  cell  masses  observed  in  the  lesions  are  composed  of 
the  proliferated  endothelial  cells  from  the  newly  formed  blood- 
vessels, and  that  these  cells  should  be  designated  as  angioblasts. 
No  giant-cells  were  found.  In  spite  of  the  superior  technique, 
and  of  employing  various  staining  methods,  which  included 
Romanowsky-Giemsa,  Gram,  and  Levaditi  methods,  as  well 
as  tubercle  bacillus  staining,  he  was  unable  to  observe  either 
bacteria  or  protozoa  in  the  lesions.  On  the  other  hand, 
within  many  endothelial  cells  as  well  as  in  the  proliferated 
angioblasts  from  the  blood-vessels,  he  found  conglomerations 
of  fine  granules  which  were  not  sharply  bounded,  and  that 
were  clearly  visible  only  in  the  sections  stained  by  Giemsa's 
and  Levaditi's  methods.  The  position,  size,  form,  and  general 
appearance  of  these  structures  reminded  the  author  greatly  of 
the  bodies  observed  in  trachoma,  molluscum  contagiosum, 
birdpox,  and  other  diseases  in  which  ''  chlamydozoa  "  have 
been  described.  No  proof  whatever  that  the  granules  Rocha- 
Lima  observed  in  the  degenerating  cells  were  parasites  is 
given.  It  seems  remarkable  that  he  should  draw  such  sweep- 
ing conclusions  regarding  the  etiology  of  this  disease  from  the 
study  of  one  case  observed  late  in  the  course  of  the  disease. 

Finally  Cole  ^  studied  histologically  the  lesions  of  a  case  of 
verruga  observed  in  Jadassohn's  clinic  in  Berne,  and  also 
those  produced  in  monkeys  by  inoculation  with  verruga 
material.  He  concludes  that  ^' as  the  other  granulomata  — 
tuberculosis,  syphilis,  sporotrichosis,  actinomycosis,  etc.,  have 
their  own  significant  histological  changes,  so  also  does  verruga 
peruviana,  belonging  to  the  same  class,  have  its  own  character- 
istic microscopical  picture.  It  is  characterized  by  a  dilatation 
of  the  lymph  vessels  and  a  choking  of  their  lumina  with  mono- 
and  polymorphonuclear  leucocytes;  also  by  an  infiltration 
around  these  vessels  of  plasma  cells,  fibroblasts,  mononuclear 

1  Cole:  Arch,  internal  rned.,  1912,  x,  668. 

J.  Cutan.  Dis.,  N.,  Y.,  1913,  xxxi,  384. 


\- 


3^' 


•  •' 


*  ■^O'^: 


^:^<# 

♦;.*  X 


r^ 


r 


.j».j^ 


■■>i   •««. 


Fig.  1.  —  Section  of  skin  lesion. 
Showing  proliferation  of  angioblasts  about  vessels. 


Fio.  2.  — ■  Very  eakly  skin  lesion.     Showing  cndotholiiil  proliferation  about  v 
Plate  XXVII.  —  Verruga  Peruviana. 


Fig.  1.  —  Early  skin  lesion.     Showing  very  vascular  structure. 


« 


:^t 


"*    t> 


M  >^  ^ 


^«l^  ^ 


*^  .1.  ^  ' 


i:"f^,"'--i 


Fic;.  2.  —  .Skin  le.sion.     Section  stained  wit  h  phosphotungstic  acid  stain 
to  show  vascuhirity. 

Platp:  XXVIII.  —  Vkhuucja  Peruviana. 


HISTOLOGICAL  STUDIES  145 

leucocytes  and  relatively  small  numbers  of  polymorphonuclear 
leucocytes.     It  is  further  characterized  by  the  formation  and 
dilatation  of  a  great  number  of  blood  capillaries  and  by  an 
extravasation  of  much  serum  and  many  red  blood  cells  into 
the  tissues.     The  Ij^mph  vessels  either  rupture  at  an  early 
stage  or  dilate  to  large  dimensions  when  their  cellular  con- 
tents undergo  a  pycnotic  degeneration  and  hyaline  change, 
with  destruction  of  the  vessel  and  invasion  of  the  mass  by 
plasma  cells  and  fibroblasts."     The  tumors  examined  from 
both  the  patient  and  the  monkeys  resembled  each  other  so 
closely  in  their  histological  structure  and  mode  of  formation, 
that  Cole  did  not  describe  them  separately.     No  giant-cells 
were  found  in  the  preparations.     Neither  in  smear  prepara- 
tions from  the  tissues  nor  in  stained  preparations  of  them 
was  any  parasite  or  organism  found  except  secondary  invad- 
ing cocci  and  bacilH.     Gram's,  Gram-Weigert's,  polychrome 
methylene    blue,    tubercle   bacillus    stains,    Mallory's   eosin, 
methylene   blue,    Mann's,    von   Krogh's  method  with  poly- 
chrome   methylene    blue,    and    Giemsa's    stain    were    used. 
Cole  does  not  refer  to  the  presence  of  "  chlamydozoa,"  but 
he  calls  particular  attention  to  large  cells  lying  in  the  center 
of  the  surviving  lymph  vessels  which  show  signs  of  degenera- 
tion.    In    these    the    chromatin    granules    are    first    massed 
together  in  the  nucleus,  then  deeply  staining  masses  are  seen 
in  the  protoplasm,  while  the  latter  begins  to  show  signs  of 
vacuolization  and  hyaline  change,  and  the  nucleus  disappears. 
Finally  the  cell  outlines  themselves  disappear  and  only  the 
masses  of  degeneration  products  and  hyaUne  are  left.     The 
endothelial  wall  about  these  large,  degenerating,  lymph  areas 
remains  intact  at  first,  but  later  disappears  when  the  sur- 
rounding fibroblasts  begin  to  penetrate  the  mass. 

Present  Investigations.  —  During  the  time  our  studies  in  Peru 
were  being  carried  on  we  collected  a  large  amount  of  perfectly 
fresh  verruga  lesions  of  the  skin  for  histological  study.  Le- 
sions in  the  different  stages  of  the  disease  from  the  very  early 
to  the  very  advanced  ones  were  obtained.  In  this  way  we 
were  able  carefully  to  study  the  manner  in  which  the  lesions 


146  VERRUGA  PERUVIANA 

develop.  The  tissues  were  hardened  in  Zenker's  solution,  in 
corrosive  sublimate  and  alcohol,  and  in  Fleming's  solution, 
or  in  formahn.  Various  stains  were  employed  including 
Wright's,  Romanowsky-Giemsa's,  Gram's,  Levaditi's,  haema- 
toxylin,  eosin,  methylene  blue  and  eosin,  Mallory's  connective 
tissue  and  phosphotungstic  haematoxylin  stains,  scharlach 
Roth,  acid  fuchsin,  and  the  tubercle  bacillus  stain. 

We  shall  now  attempt  to  give  a  general  account,  based  upon 
the  study  of  the  tumors  from  the  different  cases,  of  the  histo- 
logical appearances  of  the  lesions,  showing  the  changes  which 
they  undergo  as  they  progress. 

The  early  lesions  consist  of  newly  formed  blood-vessels  lying 
in  connective  tissue  which  as  the  lesion  progresses  in  size  be- 
comes oedematous.  The  oedematous  areas,  when  developed, 
are  often  very  poor  in  cells,  though  at  different  stages  of  the 
lesion  a  variable  number  of  lymphocytes,  larger  plasma-cells, 
and  polymorphonuclear  leukocytes  are  present.  The  early 
papule  is  rich  in  blood  and  free  blood-cells  are  usually  present. 
The  newly  formed  blood-vessels  are  often  very  numerous,  and 
a  striking  feature  in  regard  to  many  of  them  is  the  small  calibre 
of  the  vessel  in  comparison  to  the  number  and  large  amount 
of  protoplasm  present  in  their  endothelial  cells.  (Plate 
XXVII.)  In  some  instances  the  endothelial  cells  of  the  vessels 
form  more  than  one  layer,  those  on  the  outside  continuing  to 
proliferate.  Sometimes  the  appearance  suggests  that  a  cap- 
illary vessel  has  become  occluded,  and  swelling  and  prolifera- 
tion of  the  endothelial  cells  has  resulted.  The  nuclei  of  these 
cells  sometimes  show  mitosis.  As  the  lesion  progresses  there 
is  a  very  extensive  proliferation  of  these  angioblasts  which 
give  rise  to  large  islands  of  closely  placed  cells  in  which  the 
lumina  of  the  small  blood-vessels  become  compressed  and  no 
longer  visible.  Only  a  few  fibrils  can  be  detected  sometimes 
between  the  cells.  In  these  areas  and  about  their  periphery 
small  numbers  of  true  fibroblasts  may  be  made  out.  The 
prevailing  type  of  cell  in  the  early  verruga  nodule  is  the  angio- 
blast.  Other  writers  with  the  exception  of  Rocha-Lima  and 
Cole,  as  we  have  intimated,  refer  to  the  prevailing  type  of  cell 


•»% 


Fig.  1.  —  Nodule  of  Skin.     Showing  islands  of  closely  placed  angioblasts, 
oedematous  connective  tissue,  and  numerous  vessels. 


Vm.  2.  —  XoDULK  OF  Ski.n.     Showing  ishiiuls  of  angioljlusts,  oedematous  connective 
tissue,  and  numerous  vessels. 


Platk  XXIX.  —  Vkukuga  Pehuviana. 


4 


_,v.,^>     :#^-^    '^v-w   -.:    -•-'&^^/V^ 


'^y 


A 


^^A 


Fig.  1 
Section  of  nodule  showing  particularly  mitosis. 


Fig.  2 

Section  showing  proliferation  of  angioblasts, 
and  appearance  of  fibroblasts. 


Plate  XXX  —  Verruga  Peruviana 


HISTOLOGICAL   STUDIES  147 

in  the  verruga  nodule  as  the  fibroblast.     Dounon  and  Izqui- 
erdo  emphasize  the  fact  that  the  nodules  consist  of  a  structure 
resembling  sarcomatous  tissue.     Escomel  assigns   a  specific 
character  to  these  cells  and  designates  them  as  verruga  cells. 
Bindo  De  Vecchi  insists  that  they  are  merely  fibroblasts, 
while  Cole  describes  the  prevaihng  type  as  the  plasma-cell. 
A  careful  study  of  the  very  early  lesions,  however,  seems  to 
convince  one  that  these  cells  are  true  angioblasts,  a  view  also 
held  by  Rocha-Lima.     This  seems  even  more  clear  when  one 
studies  the  staining  reactions  of  these  cells  and  compares  these 
reactions  with  those  of  the  endothelial  cells  of  the  small  blood- 
vessels, and  also  studies  the  progress  of  development  of  the 
lesions.     In  no  other  condition  does  one  find  such  a  striking 
and  so  characteristic  a  proliferation  of  the  endothehal  cells 
fining  the  blood-vessels,  as  is  encountered  in  the  early  verruga 
nodule,  and  it  is  this  feature  which  particularly  distinguishes 
the  lesion  from  other  pathological  processes.     Mitotic  figures 
are  numerous  among  these  angioblasts.     (Plate  xxx.)     In  the 
older  lesions  the  fibroblasts  have  gradually  invaded  the  islands 
of  angioblasts  and  deposited  collagen  fibres  between  them. 
(Plate  xxx.)     In  this  w^ay  the  nodules  come  more  closely  to 
resemble  a  fibrosarcomatous  structure.     (Plate  xxxi.)     The 
verruga  nodule,  therefore,  constitutes  a  special  form  of  granu- 
loma characterized  in  the  early  stages  by  the  formation  of  new 
blood-vessels  in  oedematous  connective  tissue,  and  by  marked 
proliferation  of  the  angioblastic  cells  forming  masses  or  islands 
of  closely  placed  cells,  by  the  invasion  of  the  connective  tissue 
by  lymphocytes,   plasma-cells,   and  leukocytes,   and   as  the 
lesion  progresses,   by  the  formation  of  fibroblasts  and  the 
deposit  of  collagen  fibrils.     (Plates  xxvii  to  xxxii.) 

The  amount  of  connective'  tissue  varies  greatly  in  the  differ- 
ent lesions,  and  also  its  characteristics.  Sometimes  elastic 
fibers  may  be  demonstrated  by  appropriate  staining  methods. 
The  number  of  long  spindle  cells  is  greater  in  the  older  nodules, 
giving  them  more  a  fibrosarcoma-like  appearance.  The 
number  and  size  of  the  blood-vessels,  and  also  the  number  of 
free  blood-cells  varies  in  the  smaller  and  larger  lesions.     The 


148  VERRUGA  PERUVIANA 

amount  of  oedema  is  usually  greater  in  the  larger  lesions.  In 
the  subcutaneous  nodules,  however,  the  lymphocytic  infiltra- 
tion is  more  marked.  In  the  so-called  mulaire  lesions  the 
epithelium  has  often  disappeared  and  the  surface  is  covered 
with  a  more  or  less  organized  blood-clot  in  which  a  variable 
amount  of  fibrin,  polymorphonuclear  leukocytes,  and  round 
cells  are  present.  Bacteria  are  also  often  present.  Such 
lesions  beneath  the  extravasation  of  blood  often  show  a 
cavernous  structure.  In  these  areas  the  oedematous  con- 
nective tissue  is  penetrated  by  thin-walled  blood-vessels  of 
variable  size.  Often  small  extravasations  of  blood  have 
occurred  in  these  areas. 

Thus,  in  the  different  lesions  sometimes  the  condition 
found  resembles  somewhat  a  fibrosarcoma,  sometimes  a 
myxosarcoma,  and  sometimes  an  angioma.  This  seems  to 
explain  in  part  the  differences  in  the  descriptions  of  the  lesions 
which  have  been  given  by  other  investigators.  Apparently 
none  of  the  previous  histological  studies  have  been  based  upon 
a  very  large  amount  of  material.  Usually  only  a  small  amount 
of  material  from  single  cases  has  been  studied,  or  the  report 
has  been  made  from  the  study  of  a  few  pieces  of  tissue  removed 
from  a  case  which  has  occurred  at  a  distance. 

Giant-cells  were  observed  in  only  one  of  the  lesions  which  we 
have  studied.  This  was  evidently  a  very  old  lesion  in  which 
strands  and  islands  of  cornified  epidermis  were  intermingled 
with  tissue  constituting  the  nodule.  There  were  in  this  in- 
stance foreign  body  giant-cells  grouped  around  islands  of 
disintegrating  epithelium.  This  may  explain  other  observa- 
tions relative  to  the  presence  of  giant-cells  in  verruga  lesions, 
although  the  frequency  with  which  tuberculosis  has  been 
mistaken  for  verruga  has  already  been  shown.  Plasma-cells 
were  not  found  in  the  granulomatous  areas,  but  when  present 
were  seen  more  particularly  at  the  periphery  of  the  lesions  in 
some  instances  lying  in  the  oedematous  connective  tissue. 
Mast-cells  were  occasionally  observed  in  the  sections.  The 
prevaiUng  cell  in  the  cell  nests  described  above  as  constituting 
the  compact  portions  of  the  nodules,  in  the  active  phase,  as  we 


Fig.  1.  —  Section  stained  with  eosin  and  methylene  blue.     X  10. 


Fig.  2.  —  Section  stained  with  Mallohy's  connective  tissue  stain.     X  10. 
Plate  XXXI.  —  Vekkuga  Peuuviana.     Lesions  of  the  .skin. 


Fig,  1.  —  Section  stained  to  show  connective  tissue. 


Fid.  2.  —  Section  showing  cellular  structure. 
Plate  XXXII.  —  Verruga  Peruviana.    Lesions  of  tho  skin. 


HISTOLOGICAL  STUDIP:S  149 

have  emphasized,  is  the  angioblast.  These  cells  are  more  or 
less  flattened;  the  nucleus  is  large,  vesicular,  oval  in  shape, 
and  does  not  stain  deeply,  being  only  moderately  rich  in 
chromatin;  it  often  shows  mitosis.  These  cells  are  sometimes 
found  in  various  stages  of  degeneration,  and  are  often  vacuo- 
lated. They  are  the  elements  which  Escomel  refers  to  as 
''  cellules  verruqueuses,"  and  which  Cole  refers  to  as  cells  of 
the  Ijrmph  capillaries  or  plasma-cells.  Numerous  phagocytic 
endothelial  cells  sometimes  containing  red  blood-corpuscles 
and  cellular  debris  are  often  frequently  observed  in  the  tissues. 
These  were  described  by  De  Vecchi  as  the  "  globulifere  " 
cells. 

As  we  have  called  attention  to  in  other  portions  of  this 
report,  no  visible  microorganism  has  been  detected  in  prepara- 
tions made  from  the  unbroken  nodules  of  the  skin  and  in 
sections  of  the  tissues.  In  those  lesions  in  which  the  skin  had 
become  abraded  bacteria  and  a  few  blastomyces  have  been 
found  in  the  superficial  portions  of  the  lesion.  Many  degen- 
erating endothelial  cells  have  been  encountered  in  the  sections, 
the  phagocytic  cells  showing  at  times  extensive  granulation, 
and  with  nuclei  in  various  stages  of  degeneration.  Mitotic 
figures,  as  emphasized,  are  also  frequent  in  other  cells.  No 
bodies  which  definitely  could  be  identified  as  protozoa  or  other 
microorganisms  have  been  detected  in  the  cells.  The  granular 
endothelial  cells  were  much  more  commonly  encountered  in 
the  lesions  from  monkeys  than  in  those  from  human  cases. 

Lesions  in  Animals.  —  The  verruga  lesion  which  develops  as 
the  result  of  the  inoculation  by  scarification  of  the  eyebrows 
of  the  monkey  resembles  closely  that  of  the  human  being. 
The  earlier  lesions  removed  about  six  days  after  their  appear- 
ance consist  of  a  mass  of  vascular  tissue  showing  a  high  degree 
of  endothelial  proliferation.  The  blood-vessels  are  irregular 
and  tortuous  and  are  for  the  most  part  considerably  larger 
than  the  normal  capillaries.  The  endothelial  surface  of  these 
vessels  is  somewhat  irregular  from  projecting  cells.  These 
cells,  however,  are  not  wholly  confined  to  the  lining  of  the 
blood-vessels,  but  are  more  or  less  distributed  through  the 


150  VERRUGA  PERUVIANA 

tissue.  Mitotic  figures  are  numerous  among  them.  They 
frequently  show  in  the  cytoplasm  an  area  staining  with  eosin 
and  in  some  instances  this  is  large  and  prominent.  To  us, 
this  appearance  seems  to  represent  an  abnormality  in  the 
region  of  the  centrosome.  Whether  it  is  the  structure  re- 
garded by  Meyer  as  a  chlamydozoon,  is  not  known.  There  is  a 
small  amount  of  extravasated  blood  in  the  tissue  between  the 
vessels,  and  a  greater  degree  of  infiltration  with  polymorpho- 
nuclear leukocytes  than  is  usually  seen  in  the  human  lesions. 
The  skin  overlying  this  mass  of  tissue  appears  as  though  some- 
what stretched  in  the  earlier  stage  of  growth  but  otherwise 
shows  nothing  abnormal. 

Lesions  removed  about  two  weeks  after  their  first  appear- 
ance show  a  considerable  number  of  collagen  fibrils  inter- 
mingled with  the  vascular  tissue.  The  leukocytic  infiltration 
is  now  much  more  pronounced,  but  the  blood-vessels  showing 
a  thickened  and  irregular  endothelium  still  persist.  The 
overlying  epidermis  shows  ulceration  and  sometimes  marked 
changes  probably  of  a  secondary  origin  and  nature.  There 
is  marked  down-growth  of  the  epithelium  in  places  and  con- 
siderable hydropic  degeneration  with  the  production  of  micro- 
scopic vesicles.  Phagocytosis  at  this  stage  of  the  lesion  is  a 
prominent  feature,  and  cells  are  observed  distended  with 
rounded  bodies  probably  of  the  nature  of  cellular  debris.  A 
considerable  proportion  of  the  endothelial  cells  show  rounded 
hyaline  eosin  stained  inclusions. 

The  verruga  lesion  of  the  rabbit's  testis  produced  by  the 
inoculation  of  verruga  virus  shows  certain  points  in  common 
with  the  human  lesion,  but  in  the  stages  in  which  it  has  been 
observed  it  differs  more  from  this  than  does  the  lesion  of  the 
monkey.  The  lesions  are  multicentric  and  this  is  evidently 
due  to  the  manner  of  the  distribution  of  the  virus  by  being 
inoculated  in  suspension.  Proliferation  of  the  blood-vessels 
is  present,  although  it  is  a  much  less  prominent  feature  than 
is  observed  either  in  the  lesions  of  the  human  being  or  of  the 
monkey,  being  overshadowed  by  the  exudative  process  which 
is  the  most  prominent  feature.      The  central  portion  of  the 


HISTOLOGICAL   STUDIES  151 

lesion  consists  of  necrotic  tissue  infiltrated  throughout  with 
leukocytes,  a  large  proportion  of  which  are  disintegrated. 
Around  these  is  a  zone  of  vascular  tissue  through  which  the 
leukocytes  are  migrating.  At  the  periphery  of  the  lesion 
areas  are  observed  infiltrated  chiefly  with  lymphoid  and 
plasma-cells  and  with  smaller  numbers  of  polymorphonuclear 
leukocytes.  However,  the  greater  part  of  the  tissue  is  not 
composed  of  proliferating  blood-vessels  as  in  the  lesions  of 
the  monkey.  A  few  giant-cells  are  seen  but  these  are  chiefly 
around  masses  of  pigment  which  may  have  been  introduced 
with  the  inoculated  material.  An  old  lesion  removed  forty- 
seven  days  after  inoculation  and  somewhat  over  five  weeks 
after  it  first  became  palpable  shows  chiefly  foci  of  lymphoid 
cells  in  a  mass  of  fibrous  tissue.  All  evidence  of  vascular 
proliferation  has  at  this  time  disappeared. 

Hence  lesions  of  both  the  monkey  and  the  rabbit  show  a 
proliferation  of  the  blood-vessels,  but  the  lesion  of  the  monkey 
resembles  much  more  closely  the  human  one.  In  the  monkey 
there  is  considerable  infiltration  with  leukocytes  and  this 
process  increases  as  the  skin  becomes  abraded  through  the 
rubbing  of  the  lesions.  In  the  rabbit,  on  the  other  hand,  the 
cellular  infiltration  and  foci  of  necrosis  constitute  a  more 
prominent  feature  than  the  vascular  prohferation. 

Angiofibroma  Circumscriptum  Contagiosum. —  Reference  has 
been  made  to  the  disease  described  in  Brazil  by  Bassewitz 
under  the  title  of  '^  angiofibroma  cutis  circumscriptum  con- 
tagiosum," and  the  similarity  from  a  clinical  standpoint 
between  this  condition  and  verruga  peruviana  has  been  pointed 
out  on  page  136.  Histological  examination  of  preparations 
from  the  lesions  of  angiofibroma  contagiosum  have  been  made 
by  Unna,^  by  Austrogesilo,^  by  Jeanselme,^  and  by  Ben- 
necke.^  All  of  these  authors  emphasize  the  presence  of  very 
numerous  young  capillaries  in  the  tissue.  One  cannot  fail 
to  be  impressed  by  the  similarity  from  a  histological  stand- 

'  Unna:  Arch.  f.  Schiffs-  u.  Tropenhyg.,  Leipz.,  1906,  x,  204. 
2  AustroKcsilo :  ibid.,  20.5. 

*  Jean.selme:  Rev.  de  med.  et  d'hyg.  trop..  Par.,  1906,  iii,  124. 

*  Bennecke:  Arch.  f.  Schiff.s-  u.  Tropenhyg.,  Leipz.,  1906,  x,  297. 


152  VERRUGA  PERUVIANA 

point  of  these  lesions  and  those  of  verruga  peruviana,  partic- 
ularly from  the  description  of  Bennecke.  Rocha-Lima  points 
out  that  the  processes  from  the  descriptions  given  do  not  appear 
to  be  identical,  chiefly  because  in  the  telangiectatic  granu- 
lomata  the  formation  of  vessels,  and  in  the  verruga  lesion  the 
growth  of  endothelial  cells,  appears  to  be  the  most  charac- 
teristic appearance.  However,  Bennecke  emphasizes  the 
granulomatous  nature  of  the  angiofibroma  contagiosum,  the 
proliferation  and  new  formation  of  capillaries,  and  the  prolif- 
eration of  the  angioblasts.  Whether  true  fibroblasts  were 
present  he  was  not  willing  to  decide  definitely.  He  suggests 
that  the  lesions  are  of  an  infectious  nature  and  probably 
result  from  a  living  virus.  It  therefore  seems  evident  that 
from  a  histological  standpoint  as  well  as  a  clinical  one  the 
disease  angiofibroma  cutis  circumscriptum  contagiosum,  if 
not  identical,  is  probably  a  very  closely  allied  disease  to  ver- 
ruga .peruviana.  Further  observations  upon  this  question 
are,  however,  desirable  before  a  definite  conclusion  regarding 
their  identity  can  be  reached. 

Vaccination  and  Immunity 

In  Peru  the  popular  opinion  prevails  that  one  attack  of 
verruga  peruviana  confers  immunity  against  subsequent 
attacks.  It  is  certainly  exceptional  to  find  individuals  who 
have  suffered  two  distinct  attacks  of  the  disease.  We  have 
performed  experiments  in  animals  with  the  idea  of  discovering 
whether  immunity  resulted  after  inoculation  of  the  verruga 
virus.  We  have  called  attention  to  the  fact  elsewhere  in  this 
report,  that  when  the  monkey  is  inoculated  with  the  virus 
either  directly  from  man  or  from  another  monkey,  no  gen- 
eralized eruption  occurs,  but  just  as  in  the  case  when  the  calf 
is  inoculated  with  small-pox  virus,  only  a  localized  lesion 
develops.  We  have  found  that  monkeys  may  be  very  suc- 
cessfully immunized  by  a  single  cutaneous  inoculation  of  the 
verruga  virus,  and  when  such  monkeys  are  reinoculated  no 
lesion  develops.     In  all  instances  in  which  the  monkey  is 


VACCINATION   AND   IMMUNITY  153 

successfully  vaccinated  with  the  virus  in  this  manner,  it  has 
been  protected  against  a  second  attempt  at  infection.  By 
successful  vaccination  we  imply  the  development  of  the  local 
lesion  following  the  inoculation.  Animals  have  been  found 
to  be  immune  for  as  long  a  period  as  six  months  after  the 
primary  inoculation.  It  therefore  seems  probable  that  by 
using  the  virus  after  several  passages  through  monkeys,  that 
man  may  also  be  successfully  vaccinated  against  this  disease 
in  a  similar  manner  to  that  which  is  employed  against  small- 
pox. The  virus  of  verruga,  however,  so  far  has  not  been  suc- 
cessfully glycerinized,  as  we  have  called  attention  to  elsewhere. 
This  method  of  vaccination  against  verruga  seems  favorable 
since  in  one  human  case  that  was  inoculated  with  the  virus 
only  localized  lesions  developed.  It  is  hoped  that  in  the 
near  future  vaccination  may  be  given  a  careful  trial  in  Peru 
where  the  disease  is  so  prevalent. 

The  discovery  of  this  method  of  immunization  is  obviously 
very  important.  Indeed  it  seems  likely  that  the  immunity 
obtained  from  vaccination  against  verruga  may  become  as 
successful  as  vaccination  against  small-pox.  It  also  seems 
probable  that  vaccination  in  man  may  be  performed  with 
safety  as  far  as  the  disease  verruga  peruviana  is  concerned, 
as  in  small-pox,  with  virus  obtained  directly  from  the  human 
lesions.  Experiments  of  this  nature,  however,  must  be  pur- 
sued cautiously,  since  as  we  have  pointed  out  on  page  124, 
material  from  the  lesions  in  the  different  stages  of  the  disease 
seems  to  possess  a  different  infectivity,  as  evidenced  by  its 
varying  power  to  produce  lesions  in  inoculated  animals. 

Transmission  of  Verruga  Peruviana  by  Insects 

Upon  our  arrival  in  Lima  we  found  the  government  entomol- 
ogist, Charles  H.  T.  Townsend,  in  charge  of  an  investigation 
by  the  Peruvian  government  of  the  insect  transmission  of 
verruga  peruviana.  This  work  was  begun  some  time  before 
our  arrival,  and  for  several  reasons  this  subject  was  not 
directly  pursued  by  us. 


154  VERRUGA  PERUVIANA 

In  one  of  Townsend's  ^  first  articles  upon  the  subject  of 
the  insect  transmission  of  verruga  peruviana  he  states:  "  The 
Octodontidae  and  especially  the  Cricetinae,  relatives  of  the 
pocket  mice  and  grasshopper  mice,  are  very  abundant  in  forms 
in  the  Andean  region,  and  it  seems  most  probable  that  among 
them  is  to  be  found  the  primary  reservoir  of  verruga." 

"  It  seems  strongly  indicated  that  verruga  is  transmitted  by 
ticks  in  practically  the  same  manner  as  is  Rocky  Mountain 
spotted  fever  —  that  is  to  say,  that  the  early  stages  of  the  tick 
live  upon  the  small  native  mammals,  while  the  adults  attach 
to  large  animals  and  man  for  engorgement  during  which 
process  they  transmit  the  disease.  This  explains  the  mular 
eruption  in  mules  in  the  verruga  districts." 

In  March,  1913,^  he  writes  regarding  the  transmission  of  the 
disease,  "  We  may  therefore  confidently  exclude  fleas,  bed- 
bugs, hce,  and  all  other  partly  or  wholly  nocturnal  bloodsuckers 
except  ticks.  The  strictly  day-biting  bloodsuckers  —  that  is 
to  say,  those  which  never  bite  at  night,  such  as  buffalo  gnats, 
sandflies,  horseflies,  and  stable  flies  —  are  excluded  because  it 
is  certain  that  verruga  is  contracted  oftenest  at  night.  Thus, 
by  a  process  of  exclusion,  we  arrive  at  ticks  as  apparently  the 
only  possible  carrier  of  the  disease.  Most  of  the  other  blood- 
suckers are  excluded,  as  a  matter  of  fact,  because  they  bear 
little  relation  to  the  burrowing  rodent  fauna  of  the  verruga 
districts." 

It  is  notable  that  he  excludes  the  sandflies  as  transmitters 
of  the  disease.  In  July,  1913,  he  changed  his  ideas  regarding 
the  transmission  of  verruga,  and  in  a  third  article  ^  he  writes : 
"  Starting  with  the  indisputable  fact  of  the  inoculability  of  the 
disease  directly  into  the  blood,  and  the  impossibility  of  con- 
tracting it  in  any  other  way,  we  deduce  with  certainty  that  it 
is  transmitted  by  a  bloodsucker.  The  study  of  the  blood- 
sucking fauna  of  the  Rimac  verruga  zone,  carried  on  by  the 
writer  both  by  day  and  by  night,  has  to  date  disclosed  some 

^  Townsend:  J.  Economic  Entomology,  Phila.,  1913,  vi,  211. 

2  Townsend:  The  Inca  Chronicle,  Peru,  1913,  March,  v.  No.  3,  p.  14. 

3  Townsend:  Peru  To-day,  1913,  July,  v.  No.  4,  p.  840. 


traxs:missiox  by  insects  155 

fifty  species  which  have  this  habit.  These  species  may  be 
grouped  under  the  twelve  common  heads  of  inosquitos  (zan- 
cudos  or  culicids),  punkies  (chironomids),  sandflies  (phleboto- 
mids),  buffalognats  (simuHds),  horseflies  (tabanids),  stable  flies 
(stomoxids),  tickflies  (nymphiparids) , /eas,  lice,  bugs,  ticks  and 
mites.  All  of  the  above  species  have  been  found  also  outside 
the  verruga  zone  excepting  only  the  Phlebotomus,  and  several 
species  of  buffalognats  and  horseflies.  The  last  two  bite  only 
by  day  and  are  therefore  at  once  excluded  as  verruga  carriers, 
while  it  is  at  once  evident  that  all  of  those  which  extend  out- 
side the  verruga  zone  are  likewise  excluded.  This  leaves  the 
Phlebotomus  as  the  only  agent  present,  capable  of  carrying  the 
disease." 

He  concludes  this  article  with  the  statement:  "  The  entomo- 
logical evidence  altogether  against  the  Phlebotomus  as  the 
vector  of  verruga  is  so  strong  that  it  must  be  considered  amply 
sufficient  to  convict,  even  in  the  absence  of  the  transmissional 
verification." 

In  November,  1913,  he  reported  ^  upon  "  The  first  case  of 
experimental  transmission  of  verruga  accomplished  through 
the  medium  of  Phlebotomus  verrucarum  Townsend."  The  ex- 
periment is  described  as  follows : 

"  Two  hairless  dogs  of  the  species  Canis  caraibicus  (Lesson),  male  and 
female,  each  about  one  and  one-half  years  of  age,  secured  in  Chosica, 
April  24,  1913,  were  admitted  to  the  verruga  laboratory  on  that  date, 
since  which  time  neither  one  has  left  the  laboratory  at  any  time,  the  two 
having  been  kept  chained  side  by  side  continuously.  The  female  was 
used  for  the  transmission,  while  the  male  was  reserved  for  the  check. 

"  Up  to  July  10,  1913,  the  general  condition  of  the  female  had  been 
perfect.  Examination  of  her  blood  made  June  15,  showed  nothing  abnor- 
mal. The  rectal  temperature,  taken  daily  since  May  3,  varied  from  37° 
to  39°.4  C.  (98°.6  to  102°.9  F.),  with  an  average  of  38°.6  C.  (101°.5  F.). 
She  had  always  been  robust,  and  extraordinarily  full  of  animation. 

"  On  the  afternoon  of  July  11,  1913,  I  injected  the  female  subcutane- 
ously  in  the  right  shoulder  with  1  c.c.  of  artificial  serum  containing  the 
triturated  bodies  of  twenty  females  of  the  Phlebotomus,  collected  on  the 
night  of  July  9-10  in  Verrugas  Canyon,  none  of  which  showed  any  sign 
of  blood-meal  in  the  alimentary  canal. 

'  Townsend:  .J.  Am.  M.  Ass.,  Chicago,  1913,  Ixi,  No.  19,  p.  1717. 


A.M. 

38.6 

P.M. 

37.5 

(after  injection) 

39.1 

38.7 

38.7 

38.9 

38.9 

39.1 

37.7 

38.6 

156  VERRUGA   PERUVIANA 

''  The  rectal  temperatures  of  this  dog  taken  daily  at  about  11  a.m.  and 
5  P.M.,  were  as  follows: 

July  11 38.6 

12 

13 

14 

15 

16 ^ 38.6  38.35 

17 37.8  38.5 

"  These  temperatures  indicate  nothing  abnormal,  but  are  an  important 
part  of  the  clinical  history  of  the  case. 

''  July  16,  the  dog  was  noted  to  be  decidedly  thin  and  weak,  ribs  show- 
ing plainly,  very  little  appetite,  almost  entire  lack  of  animation,  and  very 
sensitive  to  handling.  Blood-smear  made  on  that  date  showed  a  limited 
number  of  endoglobular  bodies  closely  approximating  Barton's  x-bodies 
some  endoglobular  and  a  large  number  of  exoglobular  bodies  closely  similar 
to  the  x-bodies  figured  by  Darling,  extremely  numerous  remains  of 
broken-down  red  corpuscles  and  many  nucleated  reds. 

"  July  17  there  appeared  a  typical  eruption  of  the  nodular  type  on  the 
superior  surface  of  the  right  hind  foot,  the  papules  being  in  the  incipient 
stage  and  not  yet  developed  into  subspherical  nodules,  six  of  the  papules 
being  well  covered,  while  numerous  others  with  little  color  were  present 
on  both  the  right  and  left  hind  feet. 

''  On  the  morning  of  July  18,  the  six  papules  showed  deeper  color. 
Blood-smear  made  at  the  time,  from  point  well  removed  from  the  erup- 
tion, showed  the  same  very  numerous  remains  of  broken-down  reds  as 
that  of  July  16,  and  a  large  number  of  the  exoglobular  bodies  approximat- 
ing the  x-bodies  of  Darling,  but  practically  no  endoglobular  x-bodies. 

"  On  the  morning  of  July  19,  the  dog  was  still  thin  in  flesh,  but  appeared 
much  stronger  and  almost  as  animated  as  usual.  The  eruption  was  practi- 
cally the  same,  except  that  it  appeared  less  deeply  colored.  The  best 
colored  papule  was  opened  and  a  smear  made  from  it,  which  showed,  besides 
some  nucleated  reds  and  many  remains  of  broken-down  reds,  a  considerable 
number  of  bodies  bearing  a  certaiin  resemblance  to  Leishmania,  but  lacking 
the  kinetonucleus  and  manifestly  not  that  organism.  I  have  found  the 
same  type  of  bodies  in  the  Phlehotomus,  and  shall  present  description  and 
drawings  of  them  later.  The  exoglobular  x-bodies  were  also  present. 
A  smear  made  at  the  same  time,  from  a  point  removed  from  the  eruption, 
showed  remains  of  broken-down  reds,  but  not  in  such  abundance  as  July 
16  and  18. 

"  The  check  dog  to  date  has  showed  nothing  abnormal  in  either  his 
blood,  temperature,  skin  or  general  condition,  but  has  remained  absolutely 
the  same  in  all  respects.  Blood-smear  made  July  18,  showed  no  broken- 
down  reds  and  no  x-bodies  either  endoglobular  or  exoglobular. 


TRANSMISSION   BY   INSECTS  157 

"  Aside  from  this  being  the  first  experimental  demonstration  of  the 
insect  transmission  of  verruga,  the  case  is  of  great  interest  from  a  clinical 
point  of  view.  The  practical  absence  of  fever,  while  unusual,  has  been 
noted  a  good  many  times;  but  the  remarkably  short  period  elapsing  be- 
tween infection  and  eruption  claims  especial  attention." 

It  will  be  seen  from  Townsend's  account  of  this  experiment 
that  he  believes  that  by  inoculating  a  dog  with  Phlebotomus 
flies  caught  at  random  in  the  verruga  canyon,  he  produced  the 
first  or  febrile  stage  of  verruga  peruviana  in  the  animal,  found 
in  the  blood  of  this  animal  the  characteristic  parasites,  and  that 
subsequently  a  typical  eruption  of  the  nodular  type  appeared 
upon  the  right  hind  foot  of  the  animal.  The  account  of  this 
experiment  first  appeared  as  an  article  signed  by  Mr.  Town- 
send  in  one  of  the  daily  newspapers  during  our  stay  in  Peru, 
and  the  public  was  invited  to  inspect  the  dog  and  to  examine 
the  lesions  described.  We  accordingly  went  to  Chosica  and 
examined  the  animal  in  question.  Mr.  Townsend  was  away 
at  the  time,  but  his  assistant  kindly  allowed  us  to  observe  the 
dog  and  to  make  microscopical  preparations  of  its  blood.  The 
dog  appeared  active  and  fairly  healthy.  There  were  a  few 
dried  pustules  measuring  about  1  mm.  in  diameter,  and  a 
small  grayish  indefinite  mass  of  old  scar  tissue  measuring 
about  4  mm.  in  diameter  on  the  right  hind  leg  of  the  dog. 
The  lesions  shown  us  consisted  of  a  few  dried-up  pustules 
produced  perhaps  by  staphylococcus,  and  resembled  in  no 
way  the  lesions  of  verruga.  Several  specimens  of  the  dog's 
blood  were  taken,  and  these  we  stained  by  Wright's  and 
Giemsa's  stains,  and  examined  for  parasites.  No  microor- 
ganisms were  found  therein.  Subsequently  Mr.  Townsend 
brought  specimens  of  this  dog's  blood  to  the  laboratory  and 
demonstrated  to  us  the  ''  x-bodies  "  which  he  had  observed 
and  described  in  his  report.  The  red  blood-cells  in  these 
specimens  were  very  badly  crenated,  the  specimens  had  evi- 
dently been  improperly  hardened,  and  they  showed  a  very 
great  abundance  of  artefacts  and  foreign  matter.  No  bodies 
resembling  the  parasite  of  Oroya  fever  were  observed. 


158  VERRUGA  PERUVIANA 

During  the  present  year  Townsend  ^  has  again  reported 
upon  the  transmission  of  verruga  by  insects,  and  has  given  an 
account  of  an  attack  of  fever  suffered  by  Mr.  Nicholson  follow- 
ing bites  on  the  back  of  his  hands  and  wrists.  This  report 
might  at  first  sight  seem  of  considerable  importance,  and  it 
therefore  is  advisable  to  refer  to  it.  It  appears  from  the 
report  that  Mr.  Nicholson  was  bitten  by  the  flies  on  the  17th 
of  September.  To  quote  from  the  report:  "  Daily  examina- 
tion of  Mr.  Nicholson's  blood  revealed  nothing  abnormal 
until  October  1,  when  I  found  what  I  considered  to  be  the 
verruga  x-bodies  in  the  red  cells,  but  Dr.  A.  L.  Barton,  the 
best  known  authority  on  verruga,  pronounced  them  not  so. 
This  was  due  to  the  smear  having  been  somewhat  overstained 
as  compared  with  Dr.  Barton's  customary  practice  in  staining. 
These  x-bodies  continued  in  very  small  number  without 
clinical  symptoms  of  note,  other  than  a  headache  or  slight 
feverishness  at  times,  until  October  25,  when  a  decided  rise 
of  temperature  occurred  and  the  x-bodies  were  found  to  be 
much  increased  in  number.  Dr.  Barton  now  recognizes  these 
to  be  the  verruga  x-bodies." 

It  appears  from  Townsend's  second  report  that  on  Novem- 
ber 15th  the  patient's  fever  '^  subsided  for  good."  On  De- 
cember 24,  the  report  states  the  first  sign  of  eruption  appeared. 
On  December  28  and  January  8,  the  appearance  of  further 
eruption  upon  the  skin  is  referred  to.  Thus  it  would  appear 
from  the  report  that  the  eruption  described  did  not  begin  to 
appear  until  thirty-nine  days  after  Mr.  Nicholson's  fever  had 
entirely  subsided,  and  that  the  eruption  did  not  occur  until 
over  three  months  from  the  time  that  he  was  bitten  accident- 
ally through  his  mosquito  net  while  sleeping  in  a  "  Verrugas 
Canyon." 

In  another  publication  in  Science  Townsend  ^  again  reiter- 
ates his  views  regarding  the  disease  verruga  peruviana.  In  his 
most  recent  publication  upon  the  subject  ^  he  reports  another 
experiment  in  whjch  an  attempt  was  made  to  observe  the 

1  Townsend:  Entomological  News,  Phila.,  1914,  xxv.  No.  1,  p.  40;  No.  3,  p.  131. 

2  Townsend:  Science,  N.  Y.,  1914,  Jan.  16,  xxxix,  99. 

^  Townsend:  Peru  Today,  1914,  June,  vi.  No.  2,  p.  57. 


TRANSMISSION   BY   INSECTS  159 

development  of  verruga  in  a  patient  bitten  by  Phlebotomus. 
The  flies  were  brought  ahve  daily  for  two  weeks  from  Verrugas 
Canyon,  to  the  hospital  in  Callao  where  the  experiment  was 
apparently  carried  on.  "  The  patient  received  one  hundred 
and  ninetj-nine  bites,  chiefly  on  the  arms,  during  twenty-five 
hours  and  fifty-five  minutes  total  exposure  to  nine  hundred 
and  eighty-one  Phlebotomus  from  February  8  to  23,  1914." 
He  states  further:  "  The  temperature  rose  suddenly  Feb- 
ruarj^  25,  1914,  continuing  to  rise  throughout  the  following 
day,  but  fluctuating  thereafter  till  it  reached  normal  March  1, 
and  continuing  only  slightly  above  normal  if  at  all  for  two 
weeks.  It  rose  March  14,  and  fluctuated  till  the  17th;  rose 
the  20th  and  31st  of  March,  the  2d  to  5th  of  April,  and  again 
more  decidedly  the  9th  and  10th  of  April,  with  a  slight  rise  on 
the  20th  of  April  as  the  last  sign  of  the  fever.  The  blood 
showed  leucocytosis,  eosinophiles  and  the  sparse  presence  of 
bodies  which  the  writer  identifies  as  Bartonia.  No  sign  of 
malaria  was  present  in  the  blood,  nor  other  parasite  that  might 
have  produced  the  fever.  During  the  last  half  of  March 
articulation  pains  were  prominent.  The  patient  left  for 
Liverpool  June  2,  1914,  with  letters  to  Professor  Robert  New- 
stead,  of  the  Liverpool  School  of  Tropical  Medicine,  and  Dr. 
G.  H.  F.  Nuttall,  of  the  University  of  Cambridge,  asking 
that  they  examine  him  for  verruga  eruption  up  to  August  and 
later  if  possible,  photographing  any  eruption  that  may  be 
found." 

Apparently  no  further  publication  relating  to  this  patient 
has  been  made. 

Townsend  further  writes:^  "  Bacilliform  bodies  indistin- 
guishable morphologically  from  the  Bartonia  have  been  found 
by  the  writer  in  the  Phlebotomus,  in  the  lizard  blood,  in  the 
blood  of  one  guinea  pig  injected  with  the  lizard  blood,  in  the 
liver  of  the  other  guinea  pig  similarly  injected,  in  human 
verruga  eruptive  tissue;  and  not  in  rats,  burros,  dogs,  owls, 
or  doves  examined  in  Verrugas  Canyon.  The  first  guinea  pig 
injected  died  in  nine  days.  The  second  one  received  a  much 
stronger  injection  of  the  lizard  blood  and  died  in  ten  hours. 

'  Townsend;  loc.  cit. 


160  VERRUGA  PERUVIANA 

The  bodies  seem  to  he  similar  to  the  bacillus,  either  paratyphoid  B 
or  one  closely  allied  to  it,  which  Dr.  Barton  found  present  in  all 
of  the  verruga  cases  which  he  studied  from  1900  to  1909  and  with 
which  he  produced  both  fever  and  eruption  in  dogs  and  mules. 
All  of  this  indicates  the  possibility  of  the  Bartonia  being  only  a 
form  of  the  bacillus  modified  by  parasitism  of  the  ultramicro- 
scopic  verruga  organism,  and  that  the  bacillus  carries  the  actual 
infective  organisms  in  its  substance.  At  all  events  the  constant 
presence  in  verruga  blood  and  tissues  of  this  bacillus,  which 
appears  to  be  the  same  that  occurs  so  abundantly  in  the  lizard 
blood,  makes  it  practically  certain  that  it  bears  a  very  important 
relation  to  the  disease."  ^  These  statements,  it  would  appear, 
require  no  comment. 

Further  experiments  will  probably  show  whether  a  species 
of  Phlebotomus  is  the  transmitting  agent  in  Oroya  fever. 
While  we  have  some  confidence  in  Mr.  Townsend's  entomologi- 
cal work,  we  feel  from  what  we  saw  of  his  work  during  our 
stay  in  Peru  that  from  a  medical  standpoint  his  observations 
need  confirmation  before  they  can  be  accepted. 

It  seems  very  possible  that  some  arthropod  is  concerned  in 
the  transmission  of  verruga  peruviana,  but  obviously  this  fact 
has  not  as  yet  been  demonstrated,  and  it  is  also  possible  that 
the  disease  may  be  transmitted  in  a  somewhat  similar  manner 
to  small-pox.  A  modified  form  of  it  may  be  transmitted 
by  direct  inoculation,  as  has  been  shown  by  us.  Arce  ^ 
suggested  in  1889  that  verruga  might  be  transmitted  through 
the  agency  of  some  bloodsucking  insect,  but  he  referred  partic- 
ularly to  the  fiebre  grave  de  Carrion.  Plehn  ^  and  Castellani  * 
also  suggest  that  the  cause  of  the  febrile  stage  may  be  asso- 
ciated with  some  bloodsucking  animal.  As  mentioned  in 
Section  iii,  and  on  page  42,  it  seems  very  probable  that 
Oroya  fever  is  transmitted  by  some  arthropod.  Whether 
this  is  a  species  of  tick,  a  mosquito,  or  Phlebotomus,  however, 
has  not  yet  in  our  opinion  been  conclusively  demonstrated. 

^  Not  italicized  in  the  original. 
2  Arce,  Julian:  Thesis  of  Lima,  1889. 

^  Plehn:  Mense,  Handbuch  der  Tropenkrankheiten,  Leipz.,  1905,  1st  ed.,  ii,  439. 
^  Castellani:   Castellani  and  Chalmers,  Manual  of  Tropical  Medicine,  Lond.,  1913, 
2d  ed.,  1200. 


ENTOMOLOGICAL   INVESTIGATIONS  AT 
MATUCANA 

Entomological  investigations  were  made  in  Peru,  particu- 
larh^  at  Matucana,  Surco,  and  San  Bartolome,  three  towns  in 
the  '^  verruga  zone  "  along  the  Rio  Rimac.  These  towns  are 
all  stations  on  the  railroad  which  extends  from  Callao  and  Lima 
into  the  mountains  to  Oroya.  Verruga  peruviana,  Oroya 
fever,  and  malaria  occur  at  all  three  places;  Matucana  with 
an  elevation  of  7,300  feet  being  near  the  upper  limit  of  the 
"  verruga  zone,"  while  San  Bartolome  with  an  altitude  of 
about  4,000  feet  is  not  very  far  above  its  lower  limit. 

The  physiography,  climate,  and  the  flora  and  fauna  of  the 
country  about  and  between  these  towns,  especially  the  former, 
have  often  been  discussed  by  Odriozola  and  others,  and  have 
been  referred  to  on  page  58. 

During  the  week  spent  at  Matucana,  late  in  June,  the  temp- 
erature rose  daily  to  about  74°  F.  in  the  shade,  and  was  much 
higher  in  the  sunny  parts  of  the  canyon.  During  this  part 
of  the  day  there  was  a  strong  breeze  blowing  upwards.  After 
the  sun  had  set  behind  the  tops  of  the  mountains,  the  tempera- 
ture rapidly  fell  often  to  below  50°  F.  at  night,  at  which  time 
the  direction  of  the  wind  seemed  regularly  to  be  reversed. 
There  was  thus  a  great  diurnal  range  in  temperature,  from 
the  hot  day  to  the  chilly  or  even  cold  night.  In  the  shel- 
tered portions  of  the  canyon,  especially  along  some  of  the 
streams  that  flowed  down  the  lateral  canyons,  the  temperature 
did  not  rise  so  high  owing  to  the  failure  of  the  sun's  rays  to 
penetrate  into  these  places.  These  smaller  side  canyons 
have  always  been  associated  with  verruga  and  Oroya  fever, 
particularly  the  notorious  "  verrugas  canyon  "  where  these 
diseases  appeared  so  abundantly  during  the  construction  of  the 
railway  bridge  at  this  point.     (Plate  xviii,  Fig.  3,  page  59.) 


162  ENTOMOLOGICAL  STUDIES 

The  insect  fauna  is  by  no  means  meager  at  Matucana  in 
spite  of  the  extremely  dry  environment.  Beneath  the  stones 
in  the  exposed  places  were  numerous  Carabid  and  Tenebrionid 
beetles  and  a  species  of  scorpion,  Hadruroides  lunatus  Koch  is 
quite  abundant.  Most  conspicuous  along  the  trail  are  various 
bees,  of  which  a  number  of  species  were  taken.  These  have 
been  kindly  examined  by  Professor  Cockerell  who  has  already 
published  on  them  ^  and  a  list  of  the  species  is  included  in  the 
Appendix  (p.  220). 

Diptera  were  well  represented,  although  the  smaller  forms, 
like  the  acalyptrate  Muscidae  which  are  usually  associated 
with  more  humid  conditions  were  scarce  except  about  the 
water  which  is  diverted  from  the  river.  A  few  Tabanidae 
were  collected.  Among  these  were  two  large  species  probably 
of  the  genus  Pangonia,  noticeable  on  account  of  the  long,  por- 
rect  proboscis.  They  were  seen  visiting  the  flowers  of  a 
Mimosa-like  shrub,  but  also  hovered  about  us  as  if  seeking  an 
opportunity  to  bite.  The  guide  stated  that  these  flies  regu- 
larly bite  persons,  but  an  examination  of  the  alimentary  tract 
disclosed  no  blood  in  any  of  the  specimens  captured.  The 
other  species,  belonging  to  the  genus  Tabanus,  were  taken 
along  the  trail,  also  seeking  an  opportunity  to  bite  as  is  usual 
with  members  of  this  genus.  No  blood-sucking  Muscidae 
were  seen  at  Matucana,  although  Stomoxys  calcitrans  was  seen 
at  San  Bartolome.  It  is  far  more  common,  however,  at  lower 
altitudes,  particularly  in  Lima  and  Callao. 

Mosquitoes  were  very  scarce,  but  one  species  was  reared  in 
large  numbers  from  larvae  found  in  the  stagnant  water  that 
filled  two  large  concrete  pits  close  to  Matucana.  These  tanks 
are  used  as  a  supply  for  the  railroad  water  tank  at  the  Matu- 
cana railroad  station  and  were  teeming  with  larvae.  Specimens 
reared  from  these  have  been  examined  by  Dr.  H.  S.  Dyar  and 
Mr.  F.  Knab  of  the  United  States  National  Museum,  who  find 
them  to  represent  a  new  species  of  an  hitherto  undescribed  genus. 
This  species  therefore  is  of  considerable  interest,  particularly  a,s 
we  are  able  to  give  a  description  of  the  larva  and  pupa. 

1  J.  N.  Y.  Entom.  Soc,  1914,  xxii,  pp.  306-328. 


ENTOMOLOGICAL  STUDIES  163 

Phalangomyia     Dyar  and  Knab.     Adult 

"  Proboscis  long,  uniform,  nearly  straight  in  both  sexes,  in  the  male  with  a  false 
joint  near  the  middle.  Palpi  short  in  the  female;  in  the  male  long  and  slender, 
acuminate.  Antennae  fihform  in  the  female,  the  joints  with  basal  whorls  of  short 
sparse  hairs;  in  the  male  sparsely  plumose,  rather  long.  Clypeus  well  developed, 
nude. 

"  Prothoracic  lobes  small,  lateral.  jMesonotum  elongate,  convex,  with  longi- 
tucUnal  series  of  coarse  hairs  on  the  disk  and  laterally.  Scutellum  weakly  trilobe. 
Pronotum  convex,  prominent,  nude. 

"  Abdomen  subcj'hndrical  in  the  female,  truncate  at  tip;  in  the  male  long, 
slender  basalh',  depressed  outwardly. 

"  Legs  slender  throughout  and  very  long,  the  three  pairs  progressively  longer 
but  not  differing  greatly  in  length;  femora  and  tibiae  of  all  three  pairs  of  legs  of 
about  equal  length;  first  joint  of  hind  tarsi  slightly  shorter  than  the  tibia;  scraper 
of  hind  tibia  ^^ithout  complete  row  of  spines.  Claws  simple  and  small  in  the  female; 
in  the  male  large,  unequal,  and  each  with  a  single  tooth  on  the  front  and  middle 
legs. 

"  Wings  \\dth  the  venation  as  in  Culex. 

Phalangomyia  debilis     Dyar  and  Knab 

"  Female:  Proboscis  clothed  with  dark  brownish  scales.  Palpi  about  one-sixth 
as  long  as  the  proboscis,  dark  scaled,  with  a  few  coarse  bristles.  Clypeus  promi- 
nent, narrow,  rounded  anteriorly,  dark  brown.  Antennae  rather  long  and  slender, 
blackish,  with  white  rings  at  bases  of  joints;  tori  small,  yellowish.  Occiput  black- 
ish, clothed  vnth  narrow  curved  yellowish  scales,  broader  and  denser  white  ones 
along  ocular  margins,  many  erect,  very  slender,  forked  brown  scales  dorsally; 
cheeks  white  scaled. 

"  Mesonotum  dark  brown,  two  bare,  narrow,  longitudinal  lines,  the  antescutellar 
space  bare,  a  large  bare  area  laterally  on  posterior  half  of  disk;  scales  rather  sparse 
but  coarse,  narrow  curved,  yellow-brown,  larger  pale  scales  along  margins-  and  on 
posterior  half  about  the  three  bare  zones.  Scutellum  with  pale  scales,  Uke  those 
preceding,  each  lobe  with  a  large  group  of  coarse  black  bristles.  Pleurae  brown, 
with  lanceolate  white  scales. 

"  Abdomen  rather  slender,  depressed  dorsally;  dorsal  vestiture  dull  black,  the 
segments  with  very  broad,  basal  yellowish  white  bands,  the  band  on  the  second  seg- 
ment medianly  produced  to  posterior  margin,  the  white  on  the  sixth  and  seventh 
segments  occupying  more  than  the  basal  halves,  the  eighth  entirely  white  scaled; 
no  differentiated  lateral  spots;  venter  uniformly  dirty  white  scaled;  many  long, 
pale  yellomsh  bristles  at  posterior  margins  of  segments  and  ventrally,  in  appearance 
approaching  the  lateral  ciliation  present  in  many  male  mosquitoes. 

"  Legs  brownish  black,  the  tibiae  and  tarsi  with  pale  luster  beneath;  femora 
pale  beneath  to  near  apices;  knees  pale;  tibiae  narrowly  pale  at  bases,  the  tips  of 
all  three  pairs  broadly  white;  tarsi  unbanded.     Claw  formula,  0.0-0.0-0.0. 

"  Wings  hyaline,  moderately  broad;  second  marginal  cell  rather  narrow,  nearly 
three  times  as  long  as  its  petiole,  second  posterior  cell  .slightly  longer  than  its  petiole; 
scales  dark  brown,  paler,  yellowish,  at  tip  of  wing  in  a  spot  involving  tip  of  first 
vein  and  apical  halves  of  forks  of  second  vein;  outstanding  scales  long  and  linear, 
the  others  not  differentiated;  fringe  narrow,  unicolorous.  Halteres  dark,  scaled 
throughout. 

"  Length:   Body  about  5  mm.,  wing  6  mm. 


164  ENTOMOLOGICAL  STUDIES 

"Male:  Palpi  slender,  nearly  uniform,  upcurved,  exceeding  the  proboscis  by 
about  the  length  of  the  last  joint;  vestiture  brownish,  without  pale  rings,  rather 
sparse,  short,  stiff  black  hairs  at  end  of  long  joint  and  along  last  two  joints.  An- 
tennae rather  long;  last  two  joints  long,  the  others  shorter,  slender,  pale,  with 
narrow  black  rings  at  insertions  of  hairwhorls;  hairs  very  long,  moderately  abun- 
dant, brown,  shining;  tori  large,  luteous.  Coloration  similar  to  the  female.  Ab- 
domen long,  slender  near  base,  depressed  beyond;  white  dorsal  bands  broader  than 
in  the  female,  occupying  about  two-thirds  of  sixth  and  seventh  segments,  eighth 
wholly  white  scaled;  lateral  ciliation  long  and  fine,  pale  yellowish.  Wings  nearly 
as  broad  as  in  the  female;  stems  of  fork-cells  but  slightly  longer.  Claw  formula, 
1.1-1.1-0.0. 

"  Length:  Body  about  4  mm.,  wing  4  mm. 

"  Matucana,  Peru,  7,300  feet,  June- July,  1913  (C.  T.  Brues). 

Types  and  paratypes  in  the  collections  of  the  United  States  National  Museum 
and  of  Department  of  Entomology,  Harvard  School  of  Tropical  Medicine;  one 
pair  deposited  in  the  British  Museum.     Type,  No.  18361,  United  States  Nat.  Mus. 

"Male  genitalia:  Side  pieces  from  above  broadly  conical,  straight  within, 
convex  without,  about  twice  as  long  as  the  greatest  diameter,  sparsely  covered  with 
short  hairs,  a  few  long  ones  on  the  outer  side  and  a  row  of  about  seven  long  ones 
closely  crowded  in  a  line  at  the  tip  before  the  insertion  of  the  clasp  filament.  Be- 
neath the  inner  area  of  the  piece  is  cut  awaj'-  to  two-thirds  of  its  base,  but  not  any 
at  the  tip.  The  remainder  of  the  piece  forms  a  ridge,  elongate,  about  three  times  as 
long  as  wide,  deeply  excavate  on  the  inner  side  in  the  central  third,  regularly  convex 
without,  the  tip  shortly  truncate  at  the  insertion  of  the  clasp  filament;  the  inner 
angle  basally  of  the  emargination  is  densely  hairy,  with  distinct  tubercles;  beyond 
the  emargination  is  a  large  dark  chitinous  cone,  from  the  outer  aspect  of  which 
arise  (1)  a  heavy  brown  rod  which  expands  beyond  the  middle  into  a  half-disk  and 
ends  in  a  digitate  point,  and  (2)  a  long  thin  leaf-like  appendage,  hardly  widening 
outwardly,  truncate  at  tip,  its  inner  and  distal  edges  deeply  dentate,  spinose,  its 
outer  angle  prolonged  into  a  long  point.  Within  the  emargination  of  the  under 
side  of  the  side  piece  is  a  broadly  triangular  lobe,  its  outer  angle  fitting  into  and 
surpassing  the  emargination,  dark  and  densely  tubercular  and  bearing  a  brush  of 
densely  crowded  setae,  especially  long  and  dense  at  the  angle.  This  triangular 
lobe  is  joined  by  a  membrane  to  a  stout  chitinous  strip  on  the  inner  side,  at  the 
termination  of  wliich  are  two  spine-like  rods,  inserted  in  tubercles,  with  pointed, 
slightly  recurved  tips.  Clasp  filament  stout,  enlarged  a  little  at  the  base,  bent 
beyond  the  middle,  where  it  becomes  a  little  inflated  and  creased;  tip  slender, 
furcate,  with  two  widely  divergent  points.  Harpes  broad,  triangularly  rounded, 
the  inner  angle  blunt  and  bearing  a  dense  crown  of  short  spines ;  outer  angle  form- 
ing a  slender  recurved  spiral.  Harpagones  small,  plate-like,  single,  bearing  three 
or  four  stout  curved  teeth  toward  the  tip.  Unci  small,  narrow,  with  rounded  tips. 
No  basal  appendages. 

"  The  genitaha  are  plainly  of  the  Culex  type,  as  shown  by  the  presence  of  the 
prominence  bearing  a  leaf-like  appendage  and  the  tufted  harpes.  The  side  pieces 
are  specialized  and  curiously  elaborated,  but  the  undivided  harpagones  and  the 
presence  of  the  lobe  indicate  a  low  origin  for  this  form.  The  lobe  has  disappeared 
from  all  the  Culex  proper,  being  seen  only  in  low  forms  which  are  not  truly  Culex, 
such  as  C.  dyari,  C.  melanurus,  and  the  species  of  Culiseta.  In  these  the  charac- 
teristic structures  of  Culex  have  not  appeared;  but  they  are  well  shown  in  Phalan- 
gomyia." 

"  This  genus,  therefore,  forms  a  connecting  link  between  Culiseta  and  Culex, 
nearest  to  the  latter  and  modified  on  its  own  peculiar  lines." 


Fig.  1.  —  Phalangomyia  debilis 

Dyar  and  Knab. 

Full-grown  larva,  dorsal  view. 


Fig.  2.  —  Phalangomyia  debilis  Dyar 
AND  Knab.     Pupa,  lateral  view. 


Fig.  3.  —  Aphiochaeta  .scalaris  Lw. 
Lateral  view  of  male. 


Plate  XXXIII. 


ENTOMOLOGICAL  STUDIES 


165 


A  description  of  the  larva  and  a  few  sketches  illustrating 
the  more  important  anatomical  details  of  this  mosquito 
follow.     (Plate  XXXIII,  Fig.  1.) 


Phalangomyia  debilis    Dyar  &  Knab 

Larva.     Length  of  full-grown  specimens   8  to   9  mm.     Head  subtriangular, 
about  as  broad  as  long,  with  rounded  anterior  margin  and  rather  strongly  projecting 


Fig.  1.  —  Phalangomyia  debilis, 
Antenna  of  larva. 


Fig.  2.  —  Phalangomyia  debilis, 
Mandible  of  larva. 


angulations  for  the  insertion  of  the  antennae.     Antennae  (Fig.  1  Text)  with  the 
lateral  tuft  beyond  the  middle,  just  before  the  apical  third.     Mouth  brush  extensive 


Fig.  3.  —  Phalangomyia  debilis, 
Maxilla. 


Fig.  4.  —  Phalangomyia  debilis. 
Mental  plate  of  larva. 


and  rather  diffuse,  most  of  the  bristles  usually  extending  out  laterally  and  curved 
forward  in  preserved  specimens.     Mandible  (Fig.  2  Text)  with  two  stout  setae 


166  ENTOMOLOGICAL  STUDIES 

above  the  middle  of  the  external  surface  just  preceding  the  apical  fan;  apex  with 
five  teeth  of  nearly  the  same  size.  Maxilla  (Fig.  3  Text)  subovate,  one-half  longer 
than  broad,  with  one  apical  seta,  its  small  lobe  with  two  short  setae:  apical  tuft 
long,  consisting  of  delicate  bristles.  Mental  plate  (Fig.  4  Text)  with  six  teeth  on 
each  side  below  the  apical  tooth.  Head  above  with  a  transverse  series  of  tufts  of 
bristles  across  the  middle,  well  behind  the  base  of  the  antennae.  Lateral  bristles 
of  thorax  not  extending  on  to  the  disc  except  for  several  very  sparse  tufts  about 
midway  between  the  anterior  and  posterior  margins.  Abdominal  bristle  tufts 
on  the  lateral  margin  moderately  developed;  first  and  second  segments  each  with 
two  tufts  of  four  larger  and  two  smaller  bristles  respectively,  third,  fourth,  and  fifth 
each  with  a  single  tuft  of  four;  six  and  seventh  each  with  two  or  three.     In  addition 


Fig.  5.  —  Phalangomyia  debilis.      Posterior  portion  of  larva. 

there  are  on  all  the  segments  a  few  minute  hairs  or  bristles.  Seventh  segment 
without  a  dorsal  or  lateral  plate.  Lateral  comb  of  the  eighth  segment  consisting 
of  a  transversely  oval  group  of  spine-like  scales  about  25  to  30  in  number  and  not 
arranged  in  distinct  rows.  Air-tube  (Fig  5  Text)  almost  three  and  one-half  times 
as  long  as  broad  at  the  widest  place  near  the  base,  slightly  and  gradually  tapering, 
not  inflated  nor  swollen;  its  pecten  consisting  of  about  seven  very  short  spines; 
its  hairs  placed  in  four  tufts  with  sometimes  a  minute  fifth  one  in  addition.  Anal 
segment  with  the  chitinous  ring  much  narrowed  ventrally,  only  half  as  broad  there 
as  above;  anal  processes  broadly  lanceolate,  projecting  usually  about  half  the  width 
of  the  anal  ring. 

Described  from  many  specimens  taken  at  Matucana,  Peru, 
7;300  feet,  in  a  concrete  pit  containing  water  used  as  a  supply 
for  the  railroad  locomotives. 

From  a  very  large  lot,  all  alike,  only  specimens  of  P.  debilis 
were  reared,  so  that  there  is  no  possibility  of  mistake  in  asso- 
ciating the  larvae  and  adults. 

The  larva  appears  in  most  of  its  characters  to  be  closest  to 
Aedes,  with  which  it  agrees  in  the  form  of  the  respiratory 


ENTOMOLOGICAL   STUDIES  167 

siphon,  and  the  short  spine-hke  bristles  of  the  pecten  on  this 
organ.  The  ventral  narrowing  of  the  ring  on  the  anal  segment 
and  the  more  numerous  tufts  of  hairs  on  the  siphon  are  similar 
to  Culex.  From  Culiseta  it  differs  entirely  in  the  armature 
of  the  siphon.  The  dentition  of  the  mental  plate  might  be 
that  of  certain  species  of  either  Culex  or  Aedes. 

The  pupa  (Plate  xxxiii,  Fig.  2)  does  not  show  any  striking 
peculiarities. 

Larvae  of  Simulium  (Plate  xxxiv,  Fig.  2)  are  abundant  in 
the  vicinity  of  Matucana,  and  a  number  were  collected  in  a 
small  mountain  stream  that  enters  the  Rio  Rimac  just  below 
the  town.  The  season  for  adults  was  evidently  just  approach- 
ing as  none  were  seen  beneath  the  bridge  which  crosses  at 
this  point  nor  in  the  surrounding  region.  These  larvae  evi- 
dently belong  to  two  species  as  is  shown  by  the  quite  different 
conformation  of  the  labium  and  other  structures.  With  the 
larvae  were  taken  two  very  similar,  but  distinguishable  types 
of  pupae  in  addition  to  two  others  of  widely  different  appear- 
ance. These  latter  were  enclosed  in  a  cocoon  of  the  usual 
Simulium  type,  but  are  each  provided  with  such  a  peculiarly 
modified  type  of  pro-thoracic  respiratory  apparatus,  that  they 
have  been  referred  to  this  family  with  some  doubt  as  appar- 
ently no  similar  organs  have  been  described  either  in  this  or 
related  families. 

As  the  larvae  and  pupae  furnish  very  good  characters,  the 
forms  obtained  have  been  described  without  however  giving 
them  names  in  the  absence  of  adult  specimens.  A  number 
of  species  of  Simulium  are  known  from  Peru  ^  and  quite  hkely 
the  adults  of  some  of  the  forms  here  described  have  already 
been  named. 

Larva  No.  1 

Length  7  to  9  mm.  The  fans  have  about  sixty  or  sixty-five  rays.  Antennae 
with  the  second  joint  three  times  as  long  as  thick  at  the  base,  one  half  as  long  as  the 
first,  first  and  second  joints  pale  brown,  second  hyaline  at  base  and  apex;  third 
darker  brown.  The  mandibles  possess  the  apical  {)air  of  external  bristles;  the  large 
apical  teeth  and  the  two  following  are  black,  the  remainder  of  the  smaller  teeth 
light  brown.  Maxillary  palpus  without  bristles,  l)lack,  the  apex  hyaline.  Labium 
(Fig.  0  Text)  with  the  dentate;  apical  margin  rather  narrow,  the  median  tooth 
simple,  scarcely  projecting  bey(jnd  a  line  connecting  the  apices  of  the  lateral  teeth; 

■  Knab,  F.:  Simuliidae  of  Peru.      Proc.  liiol.  Soc,  Wash.,  1914,  xxvii,  i)p.  81-85. 


168  ENTOMOLOGICAL  STUDIES 

between  the  median  and  lateral  tooth  are  three  small  teeth,  the  lateral  one  of  these 
larger  than  the  others;  along  the  side  of  the  labium  is  an  oblique  close-set  row  of 
seven  bristles,  followed  by  a  smaller  pair  and  some  distance  behind  another  smaller 
one,  making  ten  in  all.  The  head  is  very  dark,  piceous  in  fully  colored  individuals; 
the  thorax  dark  gray  except  for  the  region  about  the  ventral  proleg  and  the  abdo- 


Simulium  larva,  No.  1.    Labium. 


men  is  gray  except  below  on  the  apical  half.  The  anal  respiratory  organ  is  trilobed, 
the  three  lobes  usually  simple,  sometimes  the  lateral  ones  each  with  a  small  thumb- 
shaped  secondary  lobe  at  the  base;  rarely  with  the  median  one  also  thus  divided. 
In  at  least  some  individuals  with  simple  gill-lobes  there  are  onl}^  five  bristles  on  the 
side  of  the  labium,  indicating  perhaps  a  varietal  form,  but  as  a  gill  lobe  may  some- 
times be  present  on  one  side  and  not  on  the  other  the  two  forms  are  not  very  dis- 
tinct. 

This  was  the  most  common  form  among  the  larvae  obtained. 

Larva  No.  2 

Length  10  mm.  The  fans  have  from  twenty-five  to  thirty  rays.  Antennae 
with  the  second  joint  about  four  times  as  long  as  thick  and  half  as  long  as  the  first; 
all  three  joints  pale  yellow.  Mandibles  with  the  large  apical  teeth  black,  the  others 
pale.     Maxillary  palpi  black,  not  bristly,  apex  pale.     Labium  (Fig.  7  Text)  with 


Fig.  7.  — Simulium  larva,  No.  2.     Labium. 

the  dentate  apical  margin  narrow;  median  tooth  large,  trifid  as  the  adjacent  small 
tooth  is  advanced  forward,  next  tooth  very  small,  next  larger,  followed  by  a  large 
lateral  tooth  which  is  shorter  than  the  median  one;  along  the  side  of  the  labium  is 
a  row  of  three  bristles,  the  anterior  one  large,  the  other  two  growing  successively 
smaller.  The  anal  respiratory  organs  are  not  extended  in  any  of  the  specimens 
and  I  have  been  unable  either  to  evert  or  observe  them  in  examples  treated  with 
potash  or  peroxide,  although  the  integument  covering  these  organs  is  not  destroyed 
by  such  treatment. 


Fig.  2. — ^Simulium  sp. 
Full-grown  larva.     Matucana. 


Fig.  1 .  —  CuLiciNE  Larva. 
Full-grown,  dorsal  view.     Surco. 


Plate  XXXIV. 


ENTOMOLOGICAL   STUDIES  169 

Pupa  No.  1 

Length  3.8  to  4.2  mm.  Body  entirely  enclosed  in  the  cocoon  which  projects  as 
a  rim  above  the  slightly  obUque,  truncate  anterior  surface  of  the  head.  Four 
respiratory  filaments  on  each  side,  the  main  trunk  dividing  into  two  branches  very 
near  its  base;  each  of  these  then  divides  almost  immediately,  forming  the  four 
filaments  which  do  not  subdivide  again  before  the  apex  except  rarely  in  an  occa- 
sional filament  which  divides  once  near  its  tip.  Arising  from  the  main  trunk  at  its 
extreme  base  there  are  in  addition  two  shorter  filaments  which  extend  backwards 
and  He  on  the  side  of  the  body  inside  the  cocoon.  The  third  and  fourth  segments 
of  the  abdomen  each  bear  dorsally  a  series  of  four  small  hooks  on  each  side  ventrally 
the  fifth  segment  bears  on  each  side  of  the  middle  a  pair  of  approximate  hooks; 
the  sixth  and  seventh  each  bear  the  same  number  of  hooks,  but  here  the  hooks  are 
equidistant  from  one  another,  the  pair  on  each  side  not  being  approximated. 

P^ipa  No.  2 

Length  3.8  to  4.2  mm.  Very  similar  to  the  preceding  form  and  perhaps  only  a 
variation  of  it.  The  respiratory  trunk  divides  into  two  branches  at  the  base,  one 
of  these  branches  again  divides  a  very  short  distance  beyond  and  the  other  at  double 
the  distance  from  the  base;  of  these  four  filaments  the  dorsal  one  then  later  divides 
into  two,  the  next  remains  simple.  There  are  thus  formed  six  twigs  to  the  respira- 
tory organ. 

One  specimen  of  this  form  was  taken  at  Matucana,  Peru,  at 
an  altitude  of  7,300  feet. 

Pupa  No.  3 

Length  4  mm.  Of  an  entirely  different  type  from  any  described  pupae  of 
Simuliidae  so  far  as  it  has  been  possible  to  ascertain.     Pupa  case  not  attaining  the 


Fig.  8.  —  Simulium  pupa.  No.  3.     Anterior  aspect  of  the  body. 

anterior  margin  of  the  head,  more  or  less  slipper-shaped,  but  rather  loosely  woven. 
Respiratory  organs  of  very  peculiar  form,  arising  in  the  usual  position,  but  spreading 
out  laterally  like  a  ragged  palmetto  leaf  and  lying  rather  flat,  in  a  plane  perpendicu- 
lar to  the  axis  of  the  body.  Viewed  from  the  front  the  median  margin  is  nearly 
straight  while  the  lateral  margin  though  very  irregular  is  more  or  less  semicircular. 
Each  fan  is  composed  of  eighteen  rays  connected  basally,  but  separate  apically,  at 


170  ENTOMOLOGICAL  STUDIES 

two  places  the  clefts  between  the  raj'-s  are  much  deeper,  dividing  the  rays  into  three 
groups  of  four,  six  and  eight  rays  respectively;  the  rays  are  of  nearly  the  same 
length,  except  that  the  third  ray  of  the  first  group  of  four  and  the  third  ray  of  the 
second  group  of  six  are  much  longer;  each  ray  narrows  gradually  from  the  point 
that  it  separates  from  the  adjacent  ones  till  it  becomes  extremely  thin,  from  then 
the  tip  is  prolonged  as  a  long  delicate  filament;  although  the  rays  are  continuous 
basally,  each  is  sharply  marked  off  from  its  neighbor  by  an  impressed,  punctate 
line.  Figure  8  Text  shows  the  form  and  position  of  the  respiratory  organs  as  seen 
from  directly  in  front  of  the  head,  the  eight-rayed  section  being  dorsal  in  position. 
Dorsally  the  third  and  fourth  segments  of  the  abdomen  bear  on  each  side  a  rather 
approximated  series  of  four  booklets  along  the  posterior  edge;  the  seventh  bears 
on  the  edge  a  continuous  comb-like  series  of  very  many  tiny  booklets  and  the 
eighth  four  equidistant  small  spines.  At  the  apex  of  the  abdomen  is  a  pair  of  stout^ 
upwardly  directed  claws  or  hooks.  The  ventral  segments  bear  two  small  hooks 
on  each  side  of  the  posterior  margin  of  the  fourth  and  fifth  segments,  and  one  on  each 
side  of  the  margin. 

One  specimen,  collected  at  Matucana,  Peru,  at  an  altitude 
of  7,300  feet. 

Concerning  the  affinities  of  this  peculiar  pupa  there  appears 
to  be  much  doubt,  but  it  must  undoubtedly  belong  to  the 
genus  Simulium  or  to  some  hitherto  unknown  genus.  Aside 
from  the  extraordinary  respiratory  organs,  the  cocoon,  the 
form  of  the  body  and  the  armature  of  the  abdomen  are  typi- 
cally Simuhum-like.  It  is  to  be  hoped  that  in  the  near  future 
some  one  may  be  able  to  rear  the  adult. 

Pupa  No.  4 

Length  5.5  mm.  Pupa  of  still  another  type  with  the  respiratory  organs  in  the 
form  of  balloon-shaped  sacs  (Fig.  9  Text),  one  at  each  side  of  the  thorax  in  the 


Fig.  9.  —  Simulium  pupa,  No.  4.     Ventral  view  of  anterior  portion. 


usual  position.    Viewed  from  the  ventral  side,  these  are  subtriangular  in  form,  each 
somewhat  broader  than  the  anterior  margin  of  the  body  and  narrowed  to  a  very 


INSECT   TRANSMISSION   OF   VERRUGA  171 

short,  circular  pedicel  at  the  point  of  attachment  to  the  body.  Near  the  base  each 
bears  two  much  smaller  poUiciform  projections,  one  lateral  and  the  other  ventral; 
about  the  apex  is  a  crown  of  slender  filaments,  similar  to  the  twigs  of  the  usual 
respiratory  organ,  which  marks  the  large,  convex,  more  or  less  circular  apical  surface. 
These  are  of  variable  length,  and  very  rarely  branched.  The  third  and  fourth 
dorsal  abdominal  segments  each  bear  along  the  posterior  edge  the  usual  series  of 
four  booklets  on  each  side  of  the  middle;  the  sixth  and  seventh  bear  along  the 
posterior  edge  a  comb-like  series  of  close-set  tiny  booklets,  which  become  obsolete 
laterall}'.  The  apex  of  the  abdomen  bears  two  upturned  hooks;  on  the  ventral 
side  the  fourth  segment  bears  a  tiny  booklet  each  side  of  the  middle,  the  fifth  two 
on  each  side,  the  sixth  two,  and  the  seventh  one. 

One  specimen,  taken  from  Matucana,  Peru,  at  an  altitude 
of  7,300  feet. 

Were  it  not  for  the  unusual  respiratory  organs  of  the  pre- 
ceding species,  this  one  could  not  have  been  definitely  classi- 
fied as  a  Simuliid.  It  has,  however,  the  other  characters  of 
the  group.  The  cocoon,  abdominal  hooks  and  the  microscop- 
ical structure  of  the  respiratory  filaments  are  like  that  of 
the  "  twigs  "  in  pupae  of  the  usual  type. 

Hypotheses  regarding  insect  transmission.  —  Several  hy- 
potheses have  been  advanced  concerning  possible  carriers  of 
Oroya  fever  (usually  considered  together  with  verruga  under 
the  name  of  the  latter  disease),  but  so  far  none  of  these  has 
received  actual  proof  of  its  accuracy.  These  are  very  interest- 
ing in  the  absence  of  positive  evidence  and  it  may  be  of  value 
to  review  them  briefly. 

The  character  of  the  organism  causing  Oroya  fever  suggests 
the  possibiUty  that  some  tick  may  act  as  a  carrier,  since 
Babesia,  Theileria,  Nicollia,  Nuttaha  and  Smithia  as  well  as 
Anaplasma  and  Lympho-haematocytozoa  are,  so  far  as 
known,  all  transmitted  by  ticks.  However,  no  species  of 
tick  having  habits  compatible  with  the  distribution  and  epi- 
demiology of  the  disease  has  been  discovered.  The  suggested 
similarity  of  Oroya  fever  to  the  Rocky  Mountain  spotted 
fever  of  North  America,  shown  conclusively  to  be  tick-borne, 
is  also  suggestive  of  a  tick  as  a  carrier  for  the  Peruvian 
malady.  In  view  of  lack  of  knowledge  concerning  the  or- 
ganism of  spotted  fever  the  analogy  is  far  from  complete, 
however. 


172  ENTOMOLOGICAL  STUDIES 

Among  blood-sucking  Diptera  there  are  several  families 
which  might  contain  species  fulfilling  the  requirements  of  the 
epidemiology  of  Oroya  fever. 

Diligent  search  about  Matucana  failed  to  unearth  a  single 
tick  of  any  sort  and  these  animals  are  evidently  by  no  means  of 
common  occurrence.  A  native  guide  stated  that  many  ani- 
mals were  infested  with  ticks  and  he  secured  a  number  of  these 
so-called  carrapatos  which  proved  to  be  nothing  more  than  the 
pupiparous  fly,  Melophagus  ovinus  which  is  a  common  parasite 
of  sheep  there  as  well  as  northward  in  Ecuador.  No  ticks 
could  be  secured  from  horses,  cattle  and  llamas,  which  are  said 
sometimes  to  be  infested,  so  that  it  is  impossible  to  say  whether 
the  parasites  of  these  animals  are  true  ticks  or  Hippoboscid 
flies. 

It  is  said  on  good  authority  that  there  are  no  mammals 
restricted  in  their  distribution  to  the  verruga  zones  although 
the  viscacha  {Lagidium  peruarum)  is  typical  of  these  regions 
from  which  it  extends,  however,  to  much  greater  altitudes. 
On  this  animal,  Townsend  ^  has  collected  a  Gamasid  mite 
{ILeiognathus)  and  a  tick  {Ixodes  lagotis)  not  only  on  animals 
from  the  verruga  zone  but  also  on  those  much  above  it.  Cer- 
tain mice  may  be  restricted  to  the  verruga  zones,  but  probably 
none  without  very  close  relatives  in  the  adjacent  regions. 

Townsend  has  claimed  that  the  disease  is  transmitted  by  a 
species  of  Phlebotomus.  However,  evidence  is  rather  against 
such  a  supposition,  since  as  mentioned  above  the  group  of 
organisms  to  which  Bartonella  is  related  appears  to  be  charac- 
teristically parasitic  in  the  tick.  Nevertheless  there  are 
undoubtedly  a  number  of  phlebotomic  Diptera  restricted  to  the 
verruga  zones.  We  secured  a  mosquito  at  Matucana  belonging 
to  an  undescribed  genus  {Phalangomyia)  which  is  quite  pos- 
sibly limited  to  the  Oroya  fever  zones.  Townsend  has  also 
obtained  a  species  of  Phlebotomus  at  San  Bartolome  and 
Verrugas  Canyon  which  appears  not  to  extend  beyond  the 
altitudinal  limits  of  the  zones.  Simulium  is  abundant  in  the 
Oroya  fever  zone  of  the  Rio  Rimac  from  whence  adults  have 

1  Townsend:  Bull.  Entom.  Research,  1913,  iv,  125. 


INSECT  TRANSMISSION   OF   VERRUGA  173 

been  taken  as  well  as  the  peculiar  pupae  collected  at  Matucana 
and  described  on  another  page  (167)  of  this  report.  SimuUum, 
of  course,  ordinarily  feeds  by  day  as  well  as  by  night  and  also 
occurs  just  as  commonly  in  other  regions.  Whether  the  adult 
Simuliidae  represented  by  the  highly  modified  pupae  have 
different  habits,  must  at  present  remain  an  open  question. 

The  Phlebotomus  discovered  and  named  by  Townsend 
P.  verrucarum  ^  was  thought  by  him  to  be  the  carrier  of  ver- 
ruga (including  Oroya  fever).  In  the  first  place  these  insects 
appear  only  at  night,  remaining  concealed  in  the  moist  herbage 
and  close  to  the  damp  rocks  of  the  side  canyons  during  the 
day.  In  distribution  they  are  so  far  as  has  been  ascertained 
restricted  to  the  verruga  zone,  in  fact  conditions  suitable  to 
their  development  certainly  do  not  occur  below  and  probably 
not  above  the  zone.  Thus  the  distribution  of  Phlebotomus 
as  far  as  we  know  agrees  well  with  the  geographical  range  of 
both  verruga  and  Oroya  fever  and  its  nocturnal  activity 
coincide  with  the  fact  that  at  least  Oroya  fever  is  contracted 
only  at  night.  We  have  referred  to  the  fact  on  page  154  that 
Townsend  has  reported  that  a  patient  contracted  Oroya  fever 
after  having  been  bitten  accidentally  in  ''  Verrugas  Canyon  " 
by  Phlebotomus  and  that  experiments  were  then  made  upon 
dogs  by  inoculation  with  triturated  Phlebotomus,  which  were 
claimed  to  have  later  developed  verruga  peruviana.  These 
experiments  are  not  convincing  as  we  have  shown. 

Moreover  it  will  be  seen  that  such  a  view  might  be  correct 
only  on  the  assumption  that  verruga  and  Oroya  fever  are  one 
and  the  same  disease,  or  that  Phlebotomus  is  the  carrier  of 
both  diseases  which  obviously  hardly  seems  probable.  The 
implication  of  Phlebotomus  must  therefore  be  regarded  as  we 
have  already  intimated  only  as  a  possibility  requiring  clear 
cut  experimental  results  for  its  demonstration.  Nevertheless 
this  insect  deserves  further  study,  in  connection  with  which 
it  must  naturally  be  borne  in  mind  that  there  are  undoubtedly 
many  other  insects  in  these  zones  which  remain  to  be  dis- 
covered, e.  g.,  various  mosquitoes  (Plate  xxxiv.  Fig.  1).     In 

'  Townsend:  Insec.  Inscit.  Menstruus,  1913,  i,  107. 


174  ENTOMOLOGICAL  STUDIES 

any  case  the  carrier  would  seem  to  be  a  species  of  nocturnal 
habits,  for  it  seems  probable  that  the  disease  is  contracted 
after  sunset. 

At  the  same  time  as  emphasized  elsewhere  in  this  report 
(page  160)  while  Oroya  fever  can  hardly  be  other  than  insect- 
or  tick-borne,  the  evidence  for  a  similar  method  of  transmis- 
sion for  verruga  is  far  less  complete  and  rests  entirely  on 
epidemiological  considerations.  While  these  are  very  sug- 
gestive, they  are  by  no  means  conclusive. 


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VI 

UTA 

Uta  is  a  disease  which  has  existed  in  Peru  since  prehistoric 
times,  the  lesions  of  which  have  been  supposedly  depicted  on 
the  ancient  "  huacos/'  or  pottery,  of  the  Incas.  It  has  been 
stated  in  the  earlier  literature  that  the  disease  represented 
a  form  of  syphilis,  or  of  prehistoric  leprosy,  or  a  special  form 
of  lupus  vulgaris.  Smith  ^  and  Tschudi  ^  refer  to  the  malady 
and  point  out  that  it  consists  of  an  inflammation  of  the  skin, 
endemic  in  various  parts  of  Peru,  particularly  on  the  road 
from  Lima  to  the  mines  of  Cerro  de  Pasco.  According  to 
them  the  disease  consists  of  an  inflammation  caused  by  the 
boring  of  a  parasite  through  the  skin,  frequently  of  the  scro- 
tum, the  result  being  the  production  of  an  ulcer  which  later 
takes  on  a  cancerous  or  lupous  character.  The  malady  some- 
times ends  fatally.  Hirsch  ^  states  that  uta  possibly  belongs 
in  the  group  of  diseases  caused  by  larvae  of  Oestrus,  develop- 
ing in  the  subcutaneous  tissue.  In  recent  years  two  extensive 
monographs  on  the  subject  have  appeared  from  Peru,  one  by 
Tamayo  ^  in  1908,  and  the  other  by  Palma  ^  in  1909.  Tamayo 
calls  attention  to  the  analogy  both  from  a  clinical  and  histo- 
logical standpoint  between  the  disease  and  lupus  vulgaris. 
He  however  does  not  believe  in  the  complete  identity  of  the 
two  affections,  and  thinks  that  a  final  opinion  regarding  this 
question  should  be  reserved.  His  monograph  is  accompanied 
by  numerous  photographs  illustrating  the  lesions  of  the  malady 
as  depicted  on  the  water  jars  of  the  Incas,  and  also  contains 
illustrations  of  the  early  and  late  stages  of  the  disease  as  it 
is  observed  today.     Palma  concluded  that  it  was  a  specific 

>  Smith:  Edinb.  M.  &  S.  J.,  1840,  339. 
2  Tschudi:  Oesterr.  med.  Wchnschr.,  Wien,  1846,  509. 

'  Hirsch :  Handbook  of  Geographical  and  Historical  Pathology,  Lond.,  1885,  ii,  370. 
«  Tamayo:  La  Uta  en  el  Peru,  Lima,  1908,  1;  Proc.  IV  the  Pan-American  Congress, 
Santiago  de  Chile,  Dec,  1908. 

*  Palma:  Boletin  del  Ministerio  de  Fomento,  Lima,  1908,  vi.  No.  10,  p.  1. 


176  UTA 

affection  which  was  not  to  be  confounded  with  other  South 
American  maladies,  and  that  it  was  not  a  form  of  tuberculosis. 
His  report  also  contains  many  interesting  photographs 
illustrating  various  lesions  of  the  disease.  Anderson/  who  has 
observed  uta  in  Peru,  compares  it  with  gangosa  and  states 
that  there  are  many  points  of  resemblance  between  the 
two.  He  shows  that  in  the  absence  of  syphilis,  tuberculosis, 
and  leprosy,  the  two  diseases  resemble  each  other,  but  that 
gangosa  generally  affects  the  mucous  membrane  of  the  throat 
and  pharynx  first,  in  the  shape  of  a  nodule,  which  eventually 
ulcerates,  the  ulcer  eating  through  cartilage  and  bone  and 
finally  invading  the  cheeks  and  lips.  Uta,  on  the  contrary, 
rarely  begins  anywhere  else  than  on  the  skin,  not  on  the 
mucous  membrane,  its  invasion  of  the  interior  of  the  nose 
and  mouth  being  only  a  secondary  process,  when  eventually 
the  upper  lip  and  the  nose  with  its  cartilage  may  be  eaten  away. 
The  borders  of  the  affected  part  do  not  form  a  ridge,  but 
gradually  merge  into  healthy  skin.  There  is  a  bright  red  zone 
which  fades  off  into  the  normal  color.  A  crust  forms  on  the 
ulcerating  nodule  and  in  some  cases  it  is  connected  with 
epidermic  processes  which  penetrate  to  the  deeper  layers  of 
the  cutis,  constituting  what  the  natives  call  the  '^  roots  "  or 
"  feet  "  of  the  uta. 

The  disease  was  observed  by  Past-Assistant  Surgeon  Perry, 
United  States  Public  Health  Service,  in  his  travels  through 
Peru;  and  by  Surgeon  General  Rupert  Blue  who,  in  conversa- 
tion with  one  of  us  prior  to  our  departure  from  the  United 
States,  mentioned  its  occurrence  among  the  school  children 
of  Surco,  one  of  the  Peruvian  mountain  towns  in  which  the  dis- 
ease is  very  prevalent.  In  both  Surco,  Plate  xliv.  Fig.  2,  page 
185,  and  Otao  (the  latter  town  deriving  its  name  from  the 
prevalence  of  the  malady  there)  a  large  proportion  of  the 
inhabitants  are  either  afflicted  with  the  affection,  or  show 
the  disfiguring  scars  which  have  resulted  from  a  previous 
attack,  on  the  face,  arms,  or  legs. 

1  Anderson:  Proc.  xviith  Intemat.  Cong.  Med.,  Lond.,  1913,  Sect.  21,  Trop.  Med.  & 
Hyg.,  309. 


Plate  XXXVll.^  Uta  (From  Lumiere  Plates). 
Healing  Stage  of  an  Advanced  Casf. 


CLINICAL   DESCRIPTION  177 

The  disease  begins  with  a  small,  insignificant-appearing 
papule  (Plate  xxxv)  which  gradually  increases  in  size,  and  af- 
ter a  month  or  two  a  lesion  measuring  usually  from  1  to  3  cm. 
in  diameter  is  formed.  In  this  stage  of  the  disease  the  lesion 
is  covered  by  a  more  or  less  moist,  dark  crust  from  which  a 
sticky  secretion  exudes.  On  removal  of  this  crust  a  moist, 
superficial  ulcer  which  bleeds  freely  is  revealed.  In  neglected 
cases  the  ulcerations  extend  slowly  in  size  and  in  depth,  and 
the  lesion  usually  becomes  secondarily  infected  with  bacteria 
and  sometimes  with  spirochaetae.  Larvae  of  Chrysomyia  macel- 
laria  then  sometimes  penetrate  into  the  depths  of  the  lesions, 
particularly  when  they  occur  in  the  region  of  the  nose  and 
mouth  and  pharynx.  In  such  cases  the  ulcerations  become 
very  extensive.  The  soft  and  hard  palate  may  be  destroyed  by 
the  extension  of  the  lesion,  and  the  walls  of  the  pharynx  eaten 
away.  Plate  xxxvi.  Fig.  2  illustrates  a  case  of  this  nature, 
specimens  from  which  were  obtained  through  the  assistance  of 
Dr.  W.  F.  Bailey.  In  the  great  majority  of  cases  which  are 
treated  the  ulcerations  do  not  penetrate  to  great  depth,  and 
when  the  infective  agent  is  destroyed  the  ulcer  heals  by  granula- 
tion and  a  scar  results.  (Plate  xxxvii.)  The  lesions  may  be 
single  or  multiple;  the  face,  mouth,  and  hps,  ears  and  neck  are 
more  commonly  afifected,  but  the  ulcerations  may  occur  on  the 
arms  or  legs.  (Plate  xxxvi.  Fig.  1.)  Uta  is  particularly  com- 
mon in  children.  Often  in  the  early  stages  of  the  disease  not 
a  single  papule  is  found,  but  a  group  of  them  occupying  aa 
area  of  3  or  4  cm.  in  diameter.  The  ulcers  when  they  have 
developed  do  not  have  a  punched-out  appearance  but  their 
borders  are  uneven  and  ragged.  There  is  usually  no  marked 
resemblance  between  the  lesions  of  uta  and  those  of  lupus. 
Infection  can  evidently  occur  by  direct  inoculation,  and  we 
usually  found  when  one  child  of  a  family  was  infected  the  others 
also  either  showed  the  lesions  or  the  resulting  scars.  Plate 
xxxvi,  Fig.  1  illustrates  a  mother  and  child  infected  with  the 
disease.  Whether  transmission  also  occurs  indirectly  through 
the  bite  of  some  insect,  as  seems  probable  and  as  is  popularly 
supposed,  we  are  unable  definitely  to  state. 


178  UTA 

From  the  study  of  microscopical  preparations  made  from 
the  lesions  of  uta  and  sections  of  them  we  were  able  to  show 
that  the  disease  is  due  to  a  species  of  Leishmania.  Drawings 
made  from  two  of  the  preparations,  and  a  photomicrograph 
of  a  section  from  one  of  the  lesions  are  illustrated  in  Plates 
XXXVIII  to  XL.  The  parasites  are  found  to  be  very  abundant 
in  the  early  lesions,  but  they  become  increasingly  more  diffi- 
cult to  find  as  the  lesions  increase  in  size.  Particularly  is 
this  so  after  secondary  infections  have  occurred.  The  para- 
sites are  found  sometimes  free,  but  more  often  included 
within  the  endothelial  phagocytes.  (Plate  xxxviii.)  There 
is  nothing  otherwise  particularly  characteristic  regarding 
the  histological  appearance  of  the  lesions.  They  resemble 
in  the  earlier  stages  granulation  tissue  in  which  a  large  number 
of  endothelial  and  plasma-cells  are  present.  In  cultures  made 
upon  blood  agar  from  the  lesions  the  flagellate  stage  of  the 
organism  was  obtained  and  dividing  forms  observed.  (Plate 
XXXIX,  Fig.  2.)  As  the  illustration  shows,  a  basal  granule  in 
addition  to  the  blepharoplast  is  present. 

The  classification  of  the  species  of  Leishmania  is  difficult. 
Laveran  and  Nattan-Larrier  ^  have  created  the  variety 
Leishmania  tropica  (Wright)  var.  americana  and  Vianna  the 
variety  Leishmania  hrasiliensis.  The  former  was  said  to  be 
characterized  particularly  by  the  flattened  nucleus.  For  the 
present  at  least  from  the  evidence  available  we  do  not  feel 
justified  in  creating  a  new  species  for  the  parasite  discovered 
by  us  as  the  etiological  factor  of  uta.  Attention  has  been 
called  to  the  presence  of  the  additional  basal  granule  in  the 
flagellate  stage  of  the  parasite  of  uta.  A  dog  was  inoculated 
on  August  1  upon  the  inner  surface  of  both  ears  by  a  stab  and 
scarification  with  fresh  material  from  a  lesion  upon  the  face 
of  a  case  of  uta.  On  September  16  upon  the  internal  surface 
of  the  left  ear  two  papules  measuring  3  and  4  mm.  in  diameter 
were  observed,  elevating  the  surface  of  the  skin.  (Plate  xl. 
Fig.  1.)  These  were  whitish  in  the  center  and  slightly  pink  at 
the  periphery.     In  the  right  ear  four  similar  lesions  were  also 

1  Laveran  and  Nattan-Larrier:  Bull.  Soc.  de  path,  exotique,  Par.,  1912,  v,  176,  486. 


Fig.  1 


Fig.  2 


Plate  XXXVIII  — Uta 

Preparation  made  from  a  lesion  in  the  early  stage  of  the  disease, 
showing  species  of  Leishmania,  the  etiological  factor. 


Fig.  1 

Preparation  made  from  a  lesion  in  the  early  stage  of  the  disease, 
showing  species  of  Leishmania,  the  etiological  factor. 


(^S 


^/^)  ^ 


( 


■nd 


Fig.  2 

Flagellate  Forms  of  Leishmania 
obtained  in  culture  from  an  Uta  case. 


PLATE  XXXIX  — UTA 


Fk..  1.  —  Papulak  Lesions  on  Inner  Aspect  op  Dog's  Eaks.     Appearing  about 
one  month  after  inoculation  with  material  from  early  case  of  Uta. 


Fig.  2.  —  Showing  Fkishmania  in  a  section  of  tiik  lesion. 
Plate  XL.  — Uta. 


ETIOLOGY  179 

visible,  the  largest  measuring  6X3  mm.,  the  other  three  not 
measuring  over  5X3  mm.  The  papules  were  closely  placed. 
On  September  23,  Leishmania  was  observed  for  the  first  time 
in  the  lesions.  The  parasites  were  very  difficult  to  find.  The 
lesions  were  eventually  excised  and  other  animals  inoculated, 
but  without  result. 


VII 

^  GUAYAQUIL 

Sanitary  Conditions  and  Prevailing  Diseases 

The  City  of  Guayaquil  (Plate  iii,  page  7)  is  situated  two  and 
one-half  degrees  south  of  the  equator,  on  the  north  bank  of  the 
Guayas  river,  about  forty  miles  from  its  mouth,  and  just  below 
its  formation  by  the  junction  of  the  Rio  Grande  and  Daule 
rivers.  The  land  upon  which  almost  the  entire  city  is  situated 
is  flat,  and  has  an  elevation  of  only  a  few  feet  above  the  level 
of  the  river.  At  the  extreme  right,  or  northern  end  of  the 
town,  there  is  a  low  range  of  hills  about  five  hundred  to  six 
hundred  feet  in  height,  on  the  most  eastwardly  of  which  is 
situated  the  small  barrio  of  Las  Penas.  To  the  west  of  the 
city  is  situated  another  range  of  hills  of  about  the  same  or 
slightly  greater  height,  upon  which,  however,  there  are  no 
dwellings,  but  which  is  covered  with  a  moderately  dense 
forest.  The  banks  of  the  river  on  the  opposite  side  of  the 
town  are  low,  and  covered  with  thick  tropical  verdure.  The 
population  is  estimated  at  80,000,  though  it  is  difficult  to 
believe  that  there  is  actually  so  large  a  number  of  people 
living  in  the  town.  The  city  has  been  destroyed  twice  by  fire, 
and  largely  as  a  result  the  present  streets  usually  are  of  broad 
construction.  Only  comparatively  few  of  them  are  paved 
at  aU,  and  these  have  rough,  uneven  stones.  In  many  places 
there  are  intervals  of  several  feet  between  the  stones,  and  in 
many  others  the  stones  have  become  partially  sunken,  so  that 
following  a  rain  the  water  collects  in  the  depressions.  The 
traffic  over  them  is  necessarily  much  slower  and  rougher  than 
over  the  remaining  dirt  roads.  During  the  rainy  season,  how- 
ever, many  of  the  streets  are  entirely  covered  with  water;  while 
during  the  dry  one  the  gutters  on  each  side  of  them  are  filled 
with  stagnant  water  containing  decayed  vegetable  and  animal 

180 


SANITARY  CONDITIONS  181 

matter,  and  frequently  covered  with  algae.  Open  drains 
(Plates  XLii  and  xliii)  may  be  observed  in  the  streets  of 
the  greater  part  of  the  town,  and  in  them  it  is  common  to 
find  mosquito  larvae.  The  general  hygiene  of  the  city  is  bad. 
In  the  poorer  districts  the  streets  are  usually  in  a  very  dirty 
condition.  Partially  decomposed  animals,  such  as  dogs,  cats, 
birds  and  vultures,  sometimes  may  be  seen  lying  in  the  streets, 
as  well  as  piles  of  other  decomposing  organic  material.  In 
these  portions  of  the  town  the  houses  are  without  closets, 
and  the  people  frequently  defecate  openly  in  the  streets.  In 
the  rainy  season  the  faecal  material  is  washed  away  by  the 
water,  while  in  the  dry  season  the  conditions  obviously  become 
even  more  unsanitary.  The  natives  apparently,  in  addition, 
throw  a  large  amount  of  offal  and  rubbish  from  their  houses 
into  the  streets. 

To  the  northwest  of  the  town  there  is  situated  a  large  open 
plain  several  hundred  yards  square,  and  about  four  feet  below 
the  level  of  the  streets  which  border  it.  It  is  surrounded  on 
three  sides  by  dwelling  houses,  and  on  the  fourth  the  General 
Hospital  and  cemeteries  face  it.  In  the  rainy  season  this 
plain  is  partially  under  water,  and  furnishes  an  excellent  place 
for  the  breeding  of  mosquitoes,  and  very  numerous  mosquito 
larvae  may  be  found  there.  Beneath  several  of  the  streets 
in  the  more  prosperous  portions  of  the  town  there  are  sewers 
in  which  large  numbers  of  mosquitoes  were  also  found  to  be 
breeding.  Still  further  to  the  west  of  the  city  is  a  large  estero 
which  frequently  overflows  its  banks,  and  then  covers  an 
area  of  land  of  about  one  hundred  square  yards.  About  it 
are  situated  numerous  dwelHng  houses.  Anopheles'  larvae 
were  also  encountered  in  the  shallow  water  which  surrounds 
the  houses  in  this  district.  Vultures  are  the  sanitary  scaven- 
gers of  the  city,  and  one  may  often  see  groups  of  from  twenty 
to  thirty  of  these  birds  devouring  offal  in  the  streets. 

The  majority  of  the  dweUings  in  the  poorer  districts  are  of 
bamboo  and  nipa  construction,  and  most  of  them  are  hardly 
more  than  mere  shacks.  In  the  better  districts  the  houses  are 
usually  constructed  of  wood  and  are  from  two  to  three  stories 


182  GUAYAQUIL 

in  height;  the  second  stories  project  over  the  sidewalks,  and 
furnish  a  protection  for  pedestrians  from  both  sun  and  rain. 

The  most  important  hospitals  are  the  Yellow  Fever  and 
Bubonic  Plague  Hospitals  (Plate  xli),  situated  at  the  ex- 
treme north  of  the  town,  within  fifty  yards  of  one  another; 
the  Military  Hospital,  situated  in  the  barrio  of  Las  Penas;  the 
General  Hospital;  the  Hospital  for  the  Aged  and  Infirm;  and 
the  Hospital  for  the  Insane,  all  of  which  are  situated  on  the 
northeastern  edge  of  the  town.  To  the  westward  of  the 
General  Hospital  the  large  Catholic  Cemetery  is  located,  and 
to  the  north  of  the  Yellow  Fever  Hospital  the  burying  ground 
for  the  poor. 

The  prevaihng  diseases  in  Guayaquil  are  yellow  fever, 
bubonic  plague,  dysentery,  hookworm  infection,  malaria  and 
typhoid  fever.  The  Yellow  Fever  and  Bubonic  Plague  Hos- 
pitals have  screened  wards.  Both  of  them  are  located  near 
the  banks  of  an  estero,  the  Yellow  Fever  Hospital  being  situ- 
ated between  this  estero  and  a  large  drain  (Plate  xliii,  Fig.  1), 
which  constitutes  an  excellent  breeding  place  for  mosquitoes. 
The  Yellow  Fever  Hospital  has  accommodation  for  about  one 
hundred  beds,  and  the  Plague  Hospital  for  at  least  twice  this 
number.  Both  of  these  hospitals  are  under  the  direction  of 
Dr.  Pareja,  who  very  kindly  placed  at  our  disposal  all  of  the 
material  in  them,  and  allowed  us  to  study  the  cases  clinically, 
and  to  perform  necropsies.  Fortunately  it  was  very  unusual 
to  find  a  mosquito  in  the  wards  of  the  Yellow  Fever  Hospital, 
although  flies  and  gnats  were  occasionally  encountered  there. 
However,  Stegomyia  calopus  was  not  uncommonly  observed 
about  the  Bubonic  Plague  Hospital  which,  as  has  already  been 
stated,  is  situated  at  a  distance  of  about  fifty  yards  from  it. 
In  the  Bubonic  Plague  Hospital  there  was  a  small  laboratory  in 
which  a  portion  of  our  clinical  and  laboratory  work  was  per- 
formed. On  the  occasion  of  our  first  visit  to  the  Yellow  Fever 
Hospital  we  observed  nine  cases  of  this  disease,  and  during  our 
entire  stay  fresh  cases  were  admitted  almost  every  day.  We 
were  thus  enabled  to  observe  all  the  important  clinical  fea- 
tures of  the  disease;  and  as  well  were  able  to  study  in  the  post 


Fig.  1.  —  Plague  Hospital. 


Fig.  2.  —  Yellow  Fkver  Ho.spital. 
Plate  XLI.  —  Guayaquil. 


Fig.  1.  —  Homes  of  Yellow  Fever  Immunes. 


Fifi.  2.  —  Open  Dkaix.     Bkeeding  Place  of  Mosquitoes. 


Plate  XLII.  —  Guayaquil. 


PREVAILING   DISEASES  183 

mortem  room  the  important  pathological-anatomical  changes, 
and  to  secure  ample  material  for  investigation  and  teaching 
pm*poses. 

In  the  Plague  Hospital,  on  the  occasion  of  our  first  visit,  we 
observed  about  a  dozen  cases  of  the  disease,  the  majority  of 
which  were  of  a  verj^  mild  type.  The  serum  treatment  was 
employed  in  all  of  the  cases,  a  dose  of  sixty  cubic  centimeters 
being  given  and  repeated  in  twenty-four  hours  if  the  cases  did 
not  improve.  The  mortality  from  bubonic  plague  in  Guaya- 
quil is  not  high.  Particularly  striking  is  the  great  mildness 
of  these  cases  as  compared  with  those  observed  during  epi- 
demics of  bubonic  plague  in  the  Far  East.  On  the  day  follow- 
ing our  first  visit  one  of  the  cases  died,  and  we  were  enabled 
to  perform  a  necropsy  and  to  verify  bacteriologically  the 
diagnosis  of  the  disease.  Cultures  obtained  from  the  bubo  of 
this  case,  as  weU  as  the  bubo  itself,  were  preserved.     (Plate 

XLV.) 

In  the  General  Hospital  we  were  particularly  struck  by  the 
many  very  advanced  cases  of  uncinariasis  which  we  observed 
in  the  wards.  Many  cases  of  malaria  were  also  observed 
there. 

Great  difficulty  is  experienced  in  Guayaquil  in  the  diagnosis 
of  yeUow  fever.  About  one-third  of  those  admitted  to  the 
Yellow  Fever  Hospital  were  found  by  us  to  be  suffering  from 
malarial  infection.  The  correct  differential  diagnosis  from 
malaria  upon  cUnical  grounds  is  frequently  impossible.  Ob- 
viously, however,  the  microscopical  examination  will  give 
definite  information.  On  the  other  hand,  one  must  remember 
that  in  a  city  like  Guayaquil,  where  during  the  year  about 
95  per  cent  of  the  population  are  said  to  suffer  from  malaria, 
a  concomitant  infection  with  this  disease  and  yeUow  fever  may 
likewise  exist,  and  that  even  though  malarial  parasites  may  be 
found  in  the  blood,  nevertheless  the  individual  may  likewise 
be  infected  with  yellow  fever.  We  observed  two  such  instances 
while  there. 

Guayaquil,  from  a  sanitary  standpoint,  compares  unfavor- 
ably with  some  of  the  notably  unhealthy  cities  of  the  world. 


184  GUAYAQUIL 

Milagro.  Owing  to  the  reports  that  cases  of  yellow  fever 
were  continually  appearing  in  Milagro,  it  was  decided  to  visit 
this  village,  and  to  investigate  the  sanitary  conditions  which 
prevailed  there.  Milagro  is  situated  on  the  main  railroad 
line  from  Guayaquil  to  Quito,  and  has  an  elevation  of  but 
forty-two  feet  above  sea  level.  There  are  about  6,000  inhabi- 
tants in  the  town,  and  the  number  of  deaths  varies  in  the 
rainy  season  from  six  to  ten  every  day.  The  majority  of  the 
deaths  are  said  to  be  due  to  yellow  fever  and  malaria.  Almost 
the  entire  population  is  of  the  poorer  class,  many  being  em- 
ployed upon  the  large  sugar  hacienda  situated  near  the  town. 
The  village  presents  a  forlorn  appearance;  most  of  the  houses 
are  of  cheap  nipa  construction.  A  sluggish  but  picturesque 
river  about  fifty  feet  in  width,  flows  along  the  edge  of  the  town. 
Its  shores  are  covered  with  bamboo  and  palm.  (Plate  xliv. 
Fig.  1.)  About  the  dwelling  houses  in  the  town  there  are 
numerous  barrels  and  cans  containing  water,  and  in  these  recep- 
tacles we  almost  invariably  found  large  numbers  of  mosquito 
larvae,  and  in  some  instances  the  eggs  of  Stegomyia  were  en- 
countered. Many  of  the  dwellings  were  visited  in  which  sick 
individuals  were  found,  and  we  secured  a  large  number  of  blood 
specimens.  A  large  number  of  the  people  had  enlarged 
spleens;  and  we  also  observed  many  cases  of  advanced 
anaemia  in  addition  to  the  cases  of  malaria  and  yellow  fever. 

Entomological  Investigations 

On  account  of  its  location  in  a  region  of  great  rainfall  and 
high  humidity,  nearly  at  sea  level  and  almost  on  the  equator, 
Guayaquil  and  its  environs  support  a  very  extensive  and 
varied  insect  fauna  and  in  relation  to  insect-borne  diseases 
there  are  certain  salient  features  which  attract  the  attention 
of  the  entomologist.  As  has  been  pointed  out,  the  city  fronts 
along  the  bank  of  the  Guayas  river  and  extends  as  a  quite 
thickly  settled  area  for  a  short  distance  inland.  Beyond,  the 
outlying  country  spreads  out  as  a  large  savanna  broken  here 
and  there  by  small  hills,  and  by  depressions,  many  of  which 
contain  water.     Most  of  this  water  extends  as  narrow  arms 


Fig.  1.  —  Open  Drain  behind  the  Yellow  Fever  Hospital. 


Fig.  2.  —  Breeding  Places  of  Yellow  Feveu  and  Malaria  Mosquitoes. 


Plate  XLIII.  —  Guayaquil. 


Fig.  1.  —  MiLAGRO,  near  Guayaquil.     A  yellow  fever  district. 


Fig.  2.  — Surco,  Peru,  where  Uta,  Oroya  Fever,  Malaria,  and  Verruga 
Peruvia.n'a  are  prevalent. 


Plate  XLIV. 


ENTOMOLOGICAL  INVESTIGATIONS  185 

or  bayous  and  is  ultimately  connected  with  the  Guayas 
river,  so  that  it  rises  and  falls  to  a  greater  or  less  extent  in 
conformation  to  the  tidal  ebb  and  flow  in  the  river.  About 
this  water  are  mangrove  swamps  some  of  which  hne  its  edges 
while  others  spread  out  over  more  extensive  areas.  The  region 
is  thus  a  vast  swamp  reheved  by  large  grassy  areas  and  scat- 
tered hills. 

In  spite  of  the  vast  quantities  of  stagnant  or  slowly  moving 
water,  mosquitoes  at  certain  seasons  of  the  year  are  not  nearly 
so  abundant  as  might  be  expected.  In  the  first  place  the  tidal 
water  which  forms  an  integral  part  of  the  Guayas  river  system 
is  decidedly  brackish  as  evidenced  by  its  moUuscan  and 
crustacean  fauna  as  well  as  by  the  mangroves.  During  the 
rainy  season  the  salinity  must  fall  much  lower  but  evidently 
cannot  well  support  the  larvae  of  fresh-water  insects  to  any 
considerable  extent.  During  portions  of  the  dry  season 
Anophelines  are  practically  absent,  although  a  few  larvae  are 
found  in  the  confines  of  the  city  in  broad  shallow  ditches. 
Such  is  far  from  the  condition,  however,  during  the  rainy 
months. 

Stegomyia  calopus  was  commonly  to  be  observed  in  almost 
any  part  of  the  city  during  the  day  as  well  as  in  the  evenings, 
but  in  comparison  to  various  places  in  the  southern  United 
States  and  in  the  Greater  and  Lesser  Antilles  where  this  species 
occurs,  it  could  hardly  be  considered  as  abundant.  The  larvae 
were  found  in  a  number  of  instances  breeding  in  barrels  and 
tubs  containing  water  kept  for  domestic  purposes  or  for  water- 
ing animals  within  the  city.  Owing  to  the  activities  of  the 
municipal  health  authorities,  however,  in  suppressing  such 
nuisances  the  breeding  opportunities  for  this  species  are  shghtly 
curtailed.  It  does  not  appear  that  a  temporary  practical  ehm- 
ination  of  the  yellow  fever  mosquito  from  Guayaquil  would  be 
an  extremely  difficult  undertaking.  Infected  Stegomyiae  are, 
however,  liable  to  introduction  into  the  city  at  any  time  on 
the  numerous  small  boats  or  roofed-over  rafts  which  migrate 
up  and  down  the  river  between  Guayaquil  and  the  smaller 
towns  and  settlements  further  up  the  river.     These  also  carry 


186  GUAYAQUIL 

water  and  could  at  any  time  serve  as  breeding  grounds  for 
this  mosquito. 

A  considerable  amount  of  time  was  spent  in  visiting  various 
parts  of  the  city  to  ascertain  the  opportunities  for  the  breeding 
of  Stegomyia  in  addition  to  the  aforementioned  receptacles 
in  which  water  is  kept.-  As  may  be  expected  from  the  general 
contour  of  the  region,  there  is  much  standing  water  in  the 
streets,  gutters,  open  sewers  and  scattered  isolated  pools, 
but  none  of  these  places  were  found  actually  to  harbor  Steg- 
omyia larvae,  and  the  majority  were  obviously  unsuitable  for 
this  purpose.  The  following  abstracts  from  notes  made  at  the 
time  bring  out  this  point  quite  clearly. 

'^  At  this  time,  the  18th  of  May,  the  dry  season  has  begun 
and  conditions  in  the  confines  of  the  town  are  evidently  very 
different  from  those  which  prevail  during  the  rainy  season. 
To  the  south  the  streets  are  all  very  low  and  there  is  even 
now  a  fair  amount  of  water  stagnant  in  the  streets.  This 
is  mainly  in  the  form  of  drains  which  run  out  from  the 
houses  and  also  a  few  pools  and  gutters  that  have  algae 
growing  in  them,  showing  that  they  are  of  long  standing. 
From  the  cracked  condition  of  the  clayey  surface-soil  it  is 
very  plain  that  in  the  rainy  season  there  is  practically  no 
dry  street.  At  the  present  time  mosquitoes  are  not  very 
abundant  and  this  is  readily  accounted  for  by  the  rapidity 
with  which  the  rain  disappears.  Although  it  rained  heav- 
ily two  days  ago  the  streets  are  dry  today.  In  the  paved 
street  in  front  of  the  hotel  there  are  numerous  large  depres- 
sions containing  water  due  to  the  large  size  of  the  irregular 
paving  blocks;  these  become  dry  almost  twenty-four  hours 
after  the  rain  has  ceased  and  on  account  of  traffic  cannot 
possibly  harbor  larvae  in  the  rainy  season.  The  water  from 
the  sewers  opening  into  the  street  appears  to  be  too  dirty  for 
Stegomyia  so  that  these  must  come  from  receptacles  kept  in 
or  about  the  houses." 

It  appears  that  the  opportunities  for  Stegomyia  to  breed 
are  in  many  places  limited,  but  nevertheless  this  mosquito  was 
the  commonest  species  to  be  observed  in  the  city.     In  con- 


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ENTOMOLOGICAL  INVESTIGATIONS  187 

formity  with  its  habits  as  they  have  been  observed  elsewhere, 
the  mosquitoes  were  not  numerous  during  the  day,  and  were 
observed  to  bite  almost  exchisively  in  poorly  lighted  rooms, 
very  rarely  in  a  rather  bright  light.  They  were  very  frequently 
attracted  to  a  dark  hat  if  it  was  hung  on  the  wall  of  a  room. 
They  bite  mainly  about  the  ankles  when  there  is  light.  At 
night  also  the}^  bite  most  frequently  about  the  ankles,  but  at 
that  time  about  the  neck  and  other  exposed  parts  of  the  body 
as  weU. 

This  brings  out  the  fact,  already  observed  elsewhere,  that  a 
large  Stegomyia  population  is  not  necessary  for  the  main- 
tenance of  a  constant  incidence  of  yellow  fever  in  a  community. 
The  typical  domestic  habit  of  the  species  coupled  with  the 
undoubted  great  length  of  its  adult  life  render  the  spread  of 
infection  easy  through  the  agency  of  a  small  mosquito  popula- 
tion. The  authorities  do  not  remove  cases  of  yellow  fever 
to  the  isolation  hospital  promptly  at  the  onset  of  the  disease, 
and  such  cases  though  later  screened  are  free  to  infect  many 
mosquitoes  just  at  the  period  when  such  infection  is  possible. 
Even  in  this  community  composed  mainly  of  immunes,  the 
seasonal  prevalence  of  Stegomyia  bears  a  marked  relation  to  the 
incidence  of  yellow  fever  and  it  is  evident  that  any  reduction 
of  mosquitoes  must  have  a  prompt  effect  in  lessening  the 
prevalence  of  the  fever. 

Unfortunately  we  were  not  able  to  make  any  observations 
on  fleas  in  their  relation  to  bubonic  plague.  According  to  the 
municipal  hospital  records,  this  disease  is  even  more  common 
than  yellow  fever,  with  about  the  same  mortality.  It  is 
said  to  be  spreading  rapidly  inland,  particularly  along  the 
railroad,  and  there  should  be  excellent  opportunities  for  work 
concerning  the  distribution  of  rat  fleas,  their  seasonal  prev- 
alence and  relation  to  the  dry  season.  From  data  gathered 
by  the  Servicio  de  Sanidad  Publica,^  the  minimum  incidence 
appears  to  fall  two  or  three  months  earlier  than  that  of  yellow 
fever,  that  is  to  say  from  April  to  June.  This  fact  is  very 
marked  and  is  extremely  interesting  in  connection  with  the 

'   Infonnc  Direco.  Scrv.  .S;uii(l;i(l  Piil)l.  (!u;iy:u|uil,  liUO  and  1912. 


188  GUAYAQUIL 

recent  work  of  Bacot  and  Martin  on  the  seasonal  prevalence 
of  Plague  in  India/  where  they  believe  that  the  sudden  onset 
of  the  dry  season  causes  a  great  decimation  of  fleas  through 
certain  peculiarities  of  their  feeding  habits. 

Outside  the  city  in  the  savannas  and  on  the  slopes  of  the 
hills  where  there  were  no  or  very  few  habitations  Stegomyia 
as  might  be  expected  was  practically  absent,  but  mosquitoes 
were  abundant  and  annoying.  These  which  were  almost 
exclusively  Mansonia  titillans  (Walker)  occurred  sparingly 
about  the  edges  of  the  inhabited  area,  but  were  typically 
restricted  to  the  uninhabited  areas. 

Several  other  species  were  taken  in  lesser  numbers,  as 
follows : 

Aedes  scapularis  Wied. 

Aedes  oswaldi  Lutz. 

Aedes  ?  taeniorhynchus  Wied. 

As  mentioned  above,  the  absence  of  Anophelines  was  very 
noticeable  at  that  time  of  the  year  and  contrasts  sharply  with 
their  great  abundance  during  the  rainy  season  which  was 
reported  to  us  by  several  competent  observers  who  had  lived 
in  Guayaquil  for  a  number  of  years. 

A  number  of  insects  of  other  groups  were  secured  during  our 
stay  in  Guayaquil  and  some  of  the  more  interesting  of  these 
are  reported  upon  in  the  appendix  to  this  report  on  page  209. 

1  Bacot  and  Martin:  J.  Hygiene,  Plague  Suppl.  Ill,  1914,  pp.  423-429. 


190  YELLOW  FEVER 

Yellow  Fever  Case  8.     May  22,  1913. 

Patient  an  adult,  seen  first  on  May  21,  on  the  sixth  day  of  the  disease.  Tempera- 
ture on  admission  101°.  1  F.  There  is  general  jaundice,  particularly  of  the  con- 
junctiva. The  pulse  is  slow.  The  urine  contains  much  bile  pigment;  and  on 
shaking  it  in  the  test  tube  a  foam  persists  on  the  surface  due  to  the  albumin 
which  it  contains.  The  urine  is  scanty,  less  than  600  c.c.  being  passed  in  twenty- 
four  hours;  it  contains  about  one  and  one-half  grams  of  albumin.  During  the 
following  twenty-four  hours  _the  patient  developed  marked  epigastralgia  and 
hematemesis  and  anuria  developed.  He  became  delirious,  the  temperature  fell 
to  below  normal  and  he  died  on  the  morning  of  May  22.  Microscopical  prepara- 
tions of  the  blood  and  blood  cultures  were  made  May  21. 

Autopsy.  Five  hours  post  mortem.  Conjunctivae  greenish  yellow  in  appear- 
ance. Skin  presents  a  general  yellowish  icteric  tinge.  Shght  post  mortem  lividity 
over  dependent  parts.  Rigor  mortis  well  marked.  Pupils  equal  8  mm.  in  diameter. 
Subcutaneous  fat  is  of  an  icteric  tinge.  On  opening  the  abdominal  cavity,  small 
black  punctate  hemorrhages  from  1  to  4  mm.  in  diameter,  may  be  seen  situated  in 
the  serosa  of  the  small  intestine.  They  are  sharply  circumscribed.  The  viscera 
are  all  bile-stained.  There  are  about  100  c.c.  of  greenish-tinged  clear  fluid  in  the 
abdominal  cavity. 

Heart.  The  pericardial  cavity  contains  a  small  amount  of  clear  greenish-yellow 
fluid.  There  are  no  ecchymoses  beneath  the  epicardium  or  endocardium.  The 
heart  is  of  normal  size.  Valves  all  normal.  Chambers  contain  fluid  and  partially 
clotted  blood,  together  with  some  greenish-tinged  chicken  fat  thrombi.  Heart 
muscle  pale  in  color. 

Lungs.  The  lungs  are  both  voluminous.  Left.  Base  of  the  lung  is  bound  to 
the  surface  of  the  diaphragm  by  old  adhesions.  These  are  easily  separated.  The 
anterior  surface  in  general  is  pinkish  gray  in  color,  and  the  lung  contains  air  through- 
out. At  the  apex  there  are  a  number  of  ecchymoses,  some  discrete,  1  to  3  mm. ; 
others  confluent,  forming  patches  of  about  1  cm.,  or  larger,  in  diameter,  of  a  dark 
blue  or  almost  purple  color.  Over  the  lower  lobe  there  are  also  numerous  punctate 
hemorrhages  beneath  the  pleura.  The  posterior  and  lateral  surfaces  show  diffuse 
dark  bluish  or  purple  areas,  covering  the  greater  part  of  this  portion  of  the  lung. 
The  bronchial  lymphatic  glands  at  the  base  of  the  trachea  are  dark  purple  from  the 
hemorrhages  which  have  occurred  in  them.  They  are  not  markedly  swollen. 
The  bronchi  contain  frothy  mucus.  The  mucous  membrane  is  deeply  reddened,  the 
vessels  injected.  The  cut  surface  of  the  upper  lobe  is  pinkish  red  in  color,  and 
contains  air  throughout,  except  in  a  small  calcified  area,  0.5  cm.  in  diameter,  and 
situated  4  to  5  cm.  from  the  apex.  The  lower  lobe  is  much  congested  and  very 
oedematous.  The  color  is  red.  Dark  red  or  purplish  areas,  measuring  from  2  to 
3  mm.  in  diameter  are  scattered  throughout.  Their  margins  are  irregular,  not 
round.  They  are  not  wedge-shaped,  and  the  surface  is  not  granular.  Right.  The 
right  lung  presents  in  general  the  same  appearance  as  the  left.  In  general  it  is 
pinkish  gray  in  color,  and  there  are  a  few  punctate  and  diffuse  hemorrhages  beneath 
the  pleura.  The  lung  is  voluminous,  the  surface  smooth.  The  base  is  in  general 
dark  blue  or  purple  in  color.  The  color  of  the  lower  lobe  is  more  variegated.  The 
cut  surfaces  of  the  upper  and  middle  lobes  are  in  general  normal  in  appearance. 
The  lower  lobe  shows  considerable  congestion  and  oedema,  and  has  a  mottled 
appearance,  due  to  the  hemorrhages  which  have  occurred.  The  bronchi  show  an 
appearance  similar  to  that  encountered  in  the  left  lung.  The  cut  surface  of  the 
lung  is  smooth,  and  there  are  a  few  early  hemorrhagic  infarcts. 

The  Spleen.  Measures  15.5  X  6.5  X  3  cm.  It  is  of  dull  brownish  purple  color, 
and  of  moderately  firm  consistence.     The  surface  is  in  general  smooth,  but  there 


CLINICAL   AND   PATHOLOGICAL  STUDIES  191 

are  a  few  areas  of  chronic  perisplenitis.  Cut  surface  is  dull  brown  red  mth  a 
purplish  cast.  The  follicles  are  moderatelj'  distinct  and  some  shghtlj-  swollen. 
The  trabeculae  are  not  prominent. 

The  Liver.  Measures  32  X  15.5  X  7  cm.  It  is  of  moderately  firm  consistence, 
but  tears  easily-.  The  general  color  is  dull  yellowish  brow^n.  The  surface  is 
smooth.  The  vessels  near  the  tip  of  the  left  lobe  are  prominent.  In  the  superior 
and  anterior  surfaces  of  the  right  lobe  there  is  an  area  measuring  1.5  cm.  in  diameter 
with  irregular  margin,  and  presenting  a  yellowish  peripheral  zone,  with  a  dull  dark 
brown  central  area.  The  central  portion  is  not  uniform  in  color  throughout,  but 
show^s  a  number  of  yellowish  points  1  mm.  in  diameter.  In  general  the  markings 
of  the  lobules  are  distinct.  On  cut  section  the  liver  presents  a  mottled  yellow  and 
red  appearance;  the  red  lobules  are  sometimes  surrounded  by  dull  brown  areas. 
Other  sections  show  a  coarser  red  mottling.  Still  other  areas  show  a  fine  pale 
yellowish  mottling. 

Organs  of  the  Neck.  The  tongue  shows  numerous  superficial  erosions  at  the 
edges.  The  edges  of  the  tongue  are  clean,  but  in  the  center  there  is  a  dark  red  coat. 
The  oesophagus  appears  normal  throughout.  There  is  a  large  Ascaris  lumbricoides 
lying  partially  in  the  oesophagus  and  partially  in  the  pharynx.  There  are  minute 
ecchj'moses  in  the  trachea.  The  tonsils  are  filled  with  a  dry  cheesy  material.  The 
bronchial  lymphatic  glands  are  injected,  and  of  a  dark  red  or  purple  color. 

The  Kidneys.  Measure  12.5  X  6.5  X  3.5  cm.  There  are  a  few  diffuse  hemor- 
rhages in  the  capsule  near  the  pelvis.  The  capsule  is  slightly  adherent,  and  tears 
on  removal.  The  color  of  the  surface  is  yellowish  brown.  Cortex  measures  1.4 
mm.     The  glomeruli  are  not  prominent.     The  Adrenals  appear  normal. 

The  Stomach.  Measures  25  X  15  cm.  It  is  filled  with  reddish  black  bloody 
material.  The  mucous  membrane  is  swollen  and  more  or  less  injected.  The 
duodenum  also  contains  partially  fluid  blackish  material.  The  mucous  membrane 
of  the  small  and  large  intestine  is  in  general  normal  in  appearance.  There  are 
several  Ascaris  lumbricoides  in  the  small  intestine,  and  numerous  Trichocephalus 
trichiurus  in  the  cecum.  In  the  walls  of  the  small  intestine,  beneath  the  serosa, 
there  are  numerous  punctate  (from  1  to  4  mm.  in  diameter),  black  hemorrhages. 
Portions  of  the  stomach  are  preserved  for  museum  specimens. 

The  Bladder.  The  mucous  membrane  of  the  bladder  appears  normal.  There 
is  a  small  amount  of  cloudy  urine. 

The  Brain.  The  meninges  are  slightly  injected.  The  ventricles  contain  a  small 
amount  of  clear  fluid.     The  other  organs  appear  normal. 

Bacteriology.  Cultures  were  made  from  the  blood  during  life  and  from  the 
pericardial  fluid,  heart,  spleen  and  liver,  at  autopsy,  and  microscopical  preparations 
from  the  heart,  spleen,  liver  and  kidneys. 

Histological  Examination 

Heart.     No  pathological  process  noted. 

Lung.  There  are  extensive  areas  of  hemorrhage  throughout  the  greater  portion 
of  which  the  blood  is  laked,  and  large  bacilli  are  noted  scattered  throughout  these 
portions.  There  are  here  also  small  foci  of  infiltration  with  polymorphonuclear 
leukocytes.     Many  of  these  contain  bacteria. 

Small  Intestine.  Poorly  preserved,  the  mucosa  from  the  villi  being  necrotic. 
The  section  shows  no  prominent  abnormal  feature. 

Stomach.  Few  minute  foci  infiltrated  with  polymorphonuclear  leukocj-^tes.  In 
certain  areas  a  more  diffuse  infiltration  of  lymphoid  and  plasma-cells. 

Oesophagus.     Normal. 


192  YELLOW  FEVER 

Tonsil.  Epithelium  of  the  crypts  is  infiltrated  with  leukocytes.  The  crypts 
contain  large  masses  of  bacteria  and  detritus.  In  places  there  are  defects  in  the 
epithelium  associated  with  local  necrosis.  There  are  occasional  small  hemorrhagic 
areas,  and  the  folhcles  are  to  a  large  extent  replaced  by  large,  pale  staining  cells 
many  of  wliich  are  phagocytic. 

Tongue.  Section  shows  several  small  superficial  clumps  of  organisms  which 
appear  as  tliick  rods  arranged  in  chains. 

Liver.  The  greater  portion- of  the  liver  is  necrotic  and  the  liver  parenchyma  is 
replaced  to  a  large  extent  by  blood.  The  Hving  cells  which  constitute  only  a  very 
insignificant  part  of  the  organ  are  grouped  for  the  most  part  around  the  larger 
vessels  and  are  usually  present  in  larger  amount  around  the  central  vessel  than 
aroimd  the  portal  canal.  Mitotic  figures  are  frequently  found  within  liver  cells. 
Nearly  all  cells  are  markedly  vacuolated  and  as  the  degenerated  areas  are  ap- 
proached the  cells  are  found  distended  with  vacuoles.  Intermingled  with  these 
are  red  staining  necrotic  cells  and  granular  material  representing  the  remains 
of  those  which  have  undergone  further  disintegration.  The  blood  is  in  part 
distributed  in  spaces  formerly  occupied  by  the  fiver  columns.  Although  mitotic 
figures  are  numerous,  there  is  no  evidence  of  an  arrest  of  the  destructive  process 
or  of  regeneration. 

Pancreas.  There  is  no  evidence  of  destructive  process,  but  zymogen  granules 
are  absent  throughout  the  entire  section. 

Spleen.  Lymph-follicles  are  inconspicuous.  In  several  there  are  branching 
masses  of  dense  fibrinoid  material  situated  within  their  substance.     No  pigment. 

Lymph-Node.     Contains  a  large  amount  of  carbon  pigment.     No  lesion  noted. 

Kidney.  Many  of  the  smaller  tubules  show  casts  of  hyaline  material  staining 
either  pinkish  or  bluish  with  eosin  methylene  blue.  The  glomeruli  are  considerably 
congested  as  is  also  the  greater  portion  of  the  medulla.  The  convoluted  tubules  are 
somewhat  distended;  the  epithefium  shows  no  marked  evidence  of  degeneration. 

The  Cerebrum  and  Cerebellum  show  no  lesions. 

Anatomical  Diagnosis.  Inflammatory  foci  situated  in  hemorrhagic  areas  of 
lungs.  Shght  acute  gastritis.  Slight  tonsillitis.  Ulceration  of  tongue.  Degen- 
eration and  necrosis  of  fiver  parenchjnua.  Loss  of  zymogen  in  pancreas.  Acute 
splenitis  with  fibrinoid  substance  in  follicles.     Moderate  acute  nephritis. 

Yellow  Fever  Case  20.  Woman,  aged  thirty-six  years.  Temperature  on  ad- 
mission 103°. 2  F.,  pulse  ninety-four.  There  is  practicafiy  complete  anuria. 
Marked  epigastralgia.  The  tongue  is  clean,  the  gums  reddened  and  swollen. 
There  is  marked  icterus.  The  spleen  is  not  enlarged.  The  fiver  is  painful.  There 
is  marked  metrorrhagia.  She  was  given  sodium  bicarbonate  intravenously.  The 
temperature  fell  to  below  normal  and  she  died  the  following  day.  May  23. 

Autopsy.  There  is  marked  post  mortem  rigidity.  About  3  cm.  above  and 
behind  the  right  knee  joint  there  is  a  purpfish  area  2  cm.  in  diameter.  The 
general  color  of  the  skin  is  yeUow;  the  conjunctiva  greenish  yeUow.  The  muscles 
are  normal  in  appearance,  and  of  a  duU  red  color.  The  subcutaneous  tissues  are 
bile  stained.  On  opening  the  abdominal  cavity  numerous  hemorrhages  in  the 
peritoneum  measuring  from  2  to  3  mm.  to  1  cm.  in  diameter  are  visible.  The 
vessels  in  the  serosa  of  the  transverse  colon  are  injected,  and  there  are  a  few  pin- 
point sized  hemorrhages.  There  is  also  an  extensive  hemorrhage  at  the  base  of  the 
mesentery.  The  peritoneal  cavity  contains  about  200  c.c.  of  clear  greenish  yellow 
fluid.  The  liver  projects  4  cm.  below  the  costal  margin.  There  are  some  hemor- 
rhages over  the  greater  curvature  of  the  stomach. 

Heart  is  of  about  normal  size;  contains  fluid  blood.  No  hemorrhages  in  the 
epi-  or  endocardium.    There  is  a  soldiers  spot  1  cm.  in  diameter  over  the  anterior 


CLINICAL  AND   PATHOLOGICAL  STUDIES  193 

surface  of  the  heart.  The  endocardium  has  a  shght  yellow  tinge.  The  heart 
muscle  is  paler  than  nonnal. 

Lungs.  The  lungs  are  voluminous,  and  contain  air  throughout.  There  are  old 
adhesions  over  the  posterior  surface  of  the  left  lung.  There  is  no  fluid  in  the  pleural 
cavity.  In  the  superior  lobe  of  the  left  lung  there  are  numerous  hemorrhages 
measuring  from  1  to  3  cm.  in  diameter.  The  bronchi  are  dull  red  in  color  through- 
out, but  the  bronchioles  are  normal.  The  cut  surface  is  pale  pink.  There  is  no 
oedema.  In  the  left  lung  there  are  several  small  hemorrhagic  infarcts  near  the 
apex,  6  to  8  mm.  in  diameter.  Tracheal  mucosa  is  not  greatly  reddened.  The 
bronchial  glands  are  dark  red  or  black  in  color. 

Spleen.^  The  spleen  measures  10  X  5.5  X  2.5  cm.  It  is  small  and  tears  easily • 
dark  red  in  color.  Pulp  moderate  in  amount.  Trabeculae  prominent.  Follicles 
not  visible. 

Liver.  The  Uver  is  opaque  gray  with  yellowish  green  cast,  measures  22  X  15  X 
7  cm.  There  are  a  few  small  punctate  hemorrhages,  irregular  in  outline,  scattered 
over  the  surface  from  1  to  3  mm.  in  diameter.  The  liver  tissue  is  friable  The 
lobular  margins  are  indistinct.  Cut  surface  shows  a  fine  mottled  brownish  red 
bronze.  Gall  bladder  is  distended;  diffuse  hemorrhages  in  its  wall.  The  duct  is 
patent. 

Kidneys.  The  fat  about  the  kidneys  is  greenish  yellow  in  color.  There  are  a 
few  hemorrhages  in  the  capsule  3  mm.  in  diameter.  The  left  measures  11  X  6  X 
4  cm.  Right  9.5  X  6  X  4.5  cm.  Pyramids  dark  in  color.  Cortex  7  to  8  mm. 
Glomeruh  distinctly  visible.  Capsule  tears  in  stripping;  is  slightly  adherent. 
Color  of  kidneys  bluish  red. 

Aorta  shows  no  atheroma.  The  bladder  is  contracted  and  empty.  The  mucous 
membrane  pale. 

The  Uterus.  Measures  8X5X3.5  cm.  and  contains  a  small  amount  of  clotted 
and  fluid  blood. 

The  pancreas  appears  normal. 

The  mucous  membrane  of  the  stomach  is  swollen;  reddish  gray  in  color;  and 
there  are  several  irregular  hemorrhagic  areas  not  over  1  cm.  in  diameter,'  with 
irregular  margins.  There  are  also  hemorrhages  in  the  walls  of  the  ascending  coZon. 
The  small  intestine  in  the  upper  part  is  full  of  reddish  black  fluid  material.  In  the 
cecum  and  large  intestine  the  mucous  membrane  is  grayish.  The  follicles  are  not 
swollen.     Near  the  stomach  the  mucous  membrane  shows  some  hemorrhages. 

Bacteriology.  Cultures  were  made  from  the  blood  during  fife  and  from  the  heart, 
spleen  and  hver.  Microscopical  preparations  were  also  made  from  the  blood 
during  life  and  from  the  visceral  organs  at  autopsy. 

Histological  Examination 

Heart.  No  marked  lesion  apart  from  presence  of  vacuoles  in  muscle  fibers. 
Aorta.  Negative.  Lung.  Negative.  Trachea.  Normal  except  for  hemorrhage 
in  a  small  lyrnph-node  lying  in  its  outer  wall.  Stomach.  Numerous  foci  infiltrated 
with  lymphoid  and  plasma-cells  associated  with  wliich  are  large  numbers  of  coarse 
rather  thick  bacilli  with  rounded  ends.  These,  however,  are  not  confined  to  the 
foci  of  inflammation:  the  superficial  portion  of  the  mucosa  is  markedly  congested. 
There  are  occasionally  large  masses  of  bacteria  situated  in  the  superior  portion. 

Small  Intestine.  The  villi  are  denuded  of  epithelium.  There  is  no  demonstrable 
lesion. 

Liver.  Large  proportion  of  the  liver  parenchyma  is  degenerated  or  necrotic. 
The  necrotic  cells  appear  red  and  granular.    Portions  of  the  liver  cells  also  show 


194  YELLOW  FEVER 

this  red  granular  material.  Most  of  the  liver  cells  are  greatly  enlarged  and 
vacuolated.  There  is  no  pigment  except  a  few  granules  which  are  seen  within 
liver  cells.  The  better  preserved  portions  of  the  parenchyma  show  a  more  or  less 
irregular  distribution,  occurring  both  around  the  portal  canals  and  central  vessels. 
There  is  no  marked  process  of  regeneration.  Small  foci  of  lymphoid  and  plasma- 
cell  infiltration  occur  for  the  most  part  in  the  region  of  the  portal  canal. 

Pancreas.  Certain  of  the  gland  alveoh  show  small  groups  of  degenerated  cells. 
Practically  no  zymogen  granules. 

Adrenal.  There  is  a  marked  degree  of  parenchymatous  degeneration  affecting 
chiefly  the  medulla.     There  is  considerable  congestion  present. 

Kidney.  Convoluted  tubules  somewhat  dilated  with  granular  excretion. 
Many  deeply  stained  hyaline  and  granidar  casts.  The  glomeruh  are  moderately 
injected,  and  there  is  rather  marked  injection  of  the  medulla  with  several  hemor- 
rhagic areas. 

Spleen.  Follicles  not  atrophied  and  show  no  fibrinoid  material.  The  pulp 
contains  considerable  blood.  No  pigment  present.  Large  numbers  of  phagocytic 
cells  appear  in  certain  areas.  These  contain  red  corpuscles  and  red  staining  granu- 
lar material;  they  show  only  an  occasional  granule  of  pale  pigment. 

Lymph-Node.  Peripheral  sinuses  filled  to  large  extent  with  poljmiorphonuclear 
leukocytes  and  a  small  number  of  phagocjrtic  cells.  L5Tiiph-follicles  show  small 
areas  of  degeneration  centrally  situated. 

Anatomical  Diagnosis.  Acute  parenchymatous  degeneration  of  the  liver. 
Congestion  of  gastric  mucosa.     Considerable  phagocytosis  in  spleen. 

Yellow  Fever  Case  21 .  Patient  twenty  years  of  age,  from  Riobamba.  Seen  on 
the  fourth  day  of  the  disease.  Temperature  on  entrance  103°.  2  F.  pulse  58. 
Patient  presented  marked  jaundice  of  the  skin  and  conjunctivae.  The  urine 
varied  between  900  and  300  c.c.  The  albumin  in  the  urine  varied  between  1  and 
2.5  grams.  There  was  pain  over  the  abdomen.  The  patient  developed  hema- 
temesis  and  coma,  the  pulse  rose  to  one  hundred,  and  he  died  on  the  27th  of  May. 

Cultures  were  made  from  the  blood  on  May  25th. 

Autopsy  three  and  one-half  hours  post  mortem.  Rigor  mortis  moderate. 
Livor  mortis  also  moderate.  Entire  skin  jaundiced.  Slight  hvidity  about  neck 
and  forehead.  Pupils  equal.  On  opening  the  abdominal  cavity  the  peritoneum 
is  smooth.  The  mesocoUc  lymph-nodes  are  markedly  reddened:  cavity  contains 
70  c.c.  clear  yellowish  fluid.  Bladder  distended.  There  are  two  ecchymoses  2  cm. 
in  diameter  on  the  anterior  surface.  The  pericardial  fluid  not  increased.  There 
are  greenish  yellow  chicken  fat  thrombi  in  the  heart.  Valves  normal.  Muscle 
pale  brown  in  color.  Extensive  hemorrhages  in  the  left  ventricle  beneath  the 
endocardium.  These  are  superficial,  not  invading  the  muscle,  and  measure  from 
1  to  2  cm.  in  size. 

Lungs.  Voluminous.  Pinkish  gray  anteriorly.  Left  lung,  the  surface  is 
smooth.  There  are  numerous  hemorrhages  scattered  over  the  posterior  and  lateral 
surfaces.  These  have  regular  margins,  and  measure  2  mm.  to  1.5  cm.  They  are 
dark  purple  or  black  in  color,  and  extend  into  the  depth  for  a  distance  of  about 
7  mm.  The  bronchi  are  much  reddened.  The  cut  surface  of  the  lung  is  dark  red, 
with  a  few  hemorrhagic  areas.  The  right  lung  presents  a  similar  appearance  to 
the  left.     The  bronchial  lymph-nodes  are  dark  red. 

The  Spleen  is  not  enlarged,  and  measures  11  X  7  X  3.5  cm.  The  surface  is 
smooth,  dark  blue  or  purple  in  color.  On  cut  section  the  folhcles  are  not  visible. 
The  trabeculae  may  be  distinguished.     The  cut  surface  is  dark  red  in  color. 

The  Liver.  The  surface  is  smooth;  mottled  yellowish  red  and  brown.  Cut 
surface  lobules  not  distinct.     There  is  a  red  and  greenish  yellow  mottUng.     In 


CLINICAL  AND   PATHOLOGICAL  STUDIES  195 

places  the  lobular  markings  have  an  opaque  peripheral  zone  of  a  yellowish  gray 
color,  within  wliich  is  a  reddish  ring  surrounding  a  gray  center  (pin-point  in  size). 
The  gall  bladder  is  practically  collapsed,  and  contains  a  small  amount  of  viscid 
mucoid  greenish  bile. 

The  Kidneys.  Capsules  strip  rather  easily,  but  the  surfaces  tear  sUghtly.  The 
surface  vessels  injected.  Cortex  measures  7  to  11  mm.  Dull  brownish  red  with 
yellomsh  cast.  Glomeruh  not  prominent.  Size  11.5  X  7  X  4.4  cm.  Cut  section 
reddish  yellow.  In  the  right  kidney  are  several  small  brownish  red  infarcts 
measuring  from  2  to  3  mm.  in  diameter  at  the  base.     They  are  wedge-shaped. 

The  Stomach  is  filled  with  reddish  black  blood.  There  are  round  worms  in  the 
stomach  and  oesophagus.  There  are  very  numerous  hemorrhages  in  the  mucous 
membrane  4  to  5  mm.  in  diameter. 

The  Aorta  is  smooth. 

The  Bladder  is  distended  with  urine,  wliich  is  cloudy  and  amber  colored.  The 
genital  organs  appear  normal,  as  does  the  thyroid.  Large  intestine  largely  filled 
with  bluish  black  faeces.  No  ulcerations.  No  hemorrhages.  Trichocephalus  in 
the  cecum.  There  is  dark  green  material  in  the  small  intestine,  and  no  ulcer- 
ations.    The  solitary  foUicles  are  not  prominent. 

Bacteriology.     Cultures  were  made  from  the  heart,  sjDleen  and  hver. 

Histological  Examination 

Heart.  Certain  fibers  appear  somewhat  swollen.  Nuclei  large  and  vesicular. 
Lung.  Areas  of  hemorrhage.  Stomach.  No  lesion  except  superficial  portion  of 
mucosa  somewhat  injected  in  places. 

Liver.  Changes  are  practically  identical  with  those  seen  in  Yellow  Fever  Case 
20.  Only  a  very  small  amount  of  parenchyma  takes  a  bluish  stain.  No  pigment  is 
present.  Pancreas  shows  no  lesions ;  the  glands  stain  blue  throughout;  and  show 
no  zymogen  granules.  Kidney.  Glomeruli  are  considerably  injected,  the  capsules 
somewhat  distended.  Only  an  occasional  cast  is  found  in  the  collecting  tubules. 
Testes.  Present  no  lesions.  Spermatogenesis  apparently  arrested.  Prostate.  No 
lesion.  Spleen.  FoUicles  atropliied.  Spleen  contains  a  moderate  amount  of  blood. 
Numerous  large  phagocj^tic  cells  containing  red  blood-corpuscles.     No  pigment. 

Lymph-node.  There  is  some  degeneration  and  phagocytosis  centrally  situated 
in  the  follicles. 

Anatomical  Diagnosis.  Acute  parenchymatous  degeneration  of  the  liver. 
Slight  congestion  in  the  gastric  mucosa.     Acute  nephritis. 

Yellov)  Fever  Case  26.  Patient  in  coma  May  2S.  There  is  dark  black  blood 
about  the  lips.  There  is  an  odor  of  decomposing  straw  from  the  breath.  Pulse 
is  slow,  seventy  to  the  minute.  Temperature  is  100°.  4  F.  Spleen  is  not  en- 
larged. The  conjunctivae  are  very  yellow,  and  the  face  tinged  with  j^eUow.  Pupils 
do  not  react  alike.  There  is  blood  on  the  hands  from  about  the  mouth.  The  urine 
contains  albumin.  Hematemesis  increased  and  he  gradually  sank,  and  died  May 
29. 

Microscopical  preparations  of  blood  and  cultures  taken  during  life. 

Autopsy  two  hours  after  death.  There  is  well  marked  rigiditj'  of  the  lower 
limbs,  and  moderate  rigidity  of  the  upper.  Face  has  a  distinct  icteric  tinge.  There 
is  slight  icterus  of  the  trunk.  Dried  l)lood  on  the  hands,  forearms,  thighs,  ears, 
nose  and  lips.  Livor  mortis  over  dependent  parts.  Subcutaneous  fat  moderate 
in  amount,  bile-tinged.  Muscles  of  a  good  red  color.  On  opening  the  chest  the 
lungs  are  voluminous,  the  heart  distended  with  fluid  blood.     There  are  a  few 


196  YELLOW  FEVER 

discrete  hemorrhages  in  the  mesentery,  about  1.5  to  3  mm.  in  diameter.  The 
mesenteric  glands  are  sUghtly  reddened  and  swollen.  Mesocolic  glands  more 
markedly  reddened.  No  petechiae  over  the  heart.  Pericardial  fluid  normal  and 
clear.  The  chambers  of  the  heart  contain  fluid  blood,  and  are  entirely  free  from 
clots.  No  petechiae  of  endocardium;  valves  normal  except  all  bile-stained. 
Muscle  pale  and  soft. 

Lungs.  The  lungs  are  voluminous;  anterior  surfaces  pinkish  gray  in  color. 
There  are  old  fibrinous  adhesions  on  right  side  anteriorly,  laterally  and  posteriorly. 
The  left  lung  at  the  apex  of  the  tower  lobe  shows  a  large  hemorrhagic  area  9X5  cm. 
in  diameter;  margins  irregular,  and  outside  of  this  about  one  dozen  small  hemor- 
rhages 1X5  mm.  in  diameter.  The  hemorrhages  frequently  follow  the  lobular 
markings.  At  the  apex  of  the  lung  there  are  hemorrhagic  wedge-shaped  areas. 
The  remainder  of  this  lobe,  as  well  as  the  upper  lobe,  is  entirely  free  from  hemor- 
rhages. In  the  center  of  the  large  hemorrhagic  area  on  palpation  firm  nodules 
may  be  felt  in  the  substance  of  the  lung  beneath  the  pleura.  The  right  lung,  anterior 
surface  is  entirely  free  from  hemorrhages.  Lateral  and  posterior  surface  shows  a 
large  diffuse  hemorrhage  beneath  the  pleura.  In  the  upper  lobe  the  hemorrhagic 
areas  measure  when  discrete  from  3  to  8  mm.,  and  when  confluent  several  cm.  in 
diameter.  The  middle  and  lower  lobes  show  also  numerous  discrete  and  confluent 
hemorrhagic  areas.  On  opening  the  bronchi  the  larger  ones  show  a  moderate 
injection  of  the  vessels,  and  contain  reddish  frothy  fluid.  In  the  right  lung  apart 
from  the  chronic  fibrinous  pleurisy  previously  described,  and  the  hemorrhages  in 
the  lung  substance,  the  lung  tissue  is  normal.  On  cut  section  the  lower  lobe,  and 
portion  of  the  middle  lobe,  there  is  a  large  hemorrhagic  area  which  is  found  to  extend 
in  depth  to  the  hiatus  of  the  lung,  and  indeed  to  occupy  the  greater  portion  of  the 
entire  lower  lobe.  The  lung  tissue  in  the  hemorrhagic  area  is  firm  to  the  touch, 
and  contains  but  little  air.  It  is  moderately  oedematous.  On  further  section  of 
the  lung  there  are  numerous  discrete  and  confluent  hemorrhagic  areas,  measuring 
2  to  3  mm.,  which  are  scattered  throughout  the  lung  tissue.  These  areas  have 
irregular  margins.  On  section  of  the  left  lung  through  the  hemorrhagic  areas  the 
hemorrhages  are  found  to  extend  into  the  lung  tissue  for  a  depth  of  2  to  3  cm.  The 
more  nodular  areas  which  may  be  felt  beneath  the  pleura,  as  one  passes  the  hand 
over  the  surface,  are  found  on  section  to  consist  of  distinct  hemorrhagic  infarcts, 
and  over  these  nodules  of  infarction  the  pleura  is  arched.  (Sections  through  the 
hemorrhagic  infarcts  saved  in  Zenker's  solution.)  Otherwise  the  lung  appears  nor- 
mal; tissue  everywhere  containing  air,  and  is  not  congested  or  oedematous.  The 
bronchial  lymphatic  glands  at  the  base  of  the  trachea  are  black  from  the  -resulting 
hemorrhages  which  have  occurred  in  the  gland. 

Spleen.  The  spleen  is  of  about  normal  size,  measures  13  X  8  X  3  cm.  Sur- 
face smooth,  dark  purple  in  color.  On  cut  section  the  pulp  is  rather  soft,  and  the 
follicles  indistinctly  visible.     The  trabeculae  are  visible  in  places. 

Kidneys.  The  capsules  strip  with  shght  difficulty,  tearing  on  separation.  The 
stellate  veins  are  injected,  and  form  a  marked  contrast  in  color  to  the  distinctly 
yeUow  fatty  cortex.  There  is  practically  no  red  color  in  the  cortex.  On  cut 
section  the  cortex  measures  from  7  or  8  mm.  to  1  cm.  in  thickness.  The  surface  of 
the  cut  section  is  walnut  colored.  The  vessels  of  the  pyramids  are  moderately 
injected.  The  glomeruU  are  not  visible.  The  vessels  of  the  pelvis  are  injected. 
Adrenals  are  normal. 

Liver.  Surface  is  smooth,  consistence  is  soft,  and  tears  rather  easily.  Surface 
presents  a  mottled  appearance.  In  general  it  is  brownish  red  and  yellowish.  The 
reddish  areas  are  apparently  due  to  the  dilated  vessels.  There  are  a  few  yellowish 
areas  beneath  the  surface  of  the  capsule,  measuring  1  to  2  mm.  in  diameter.     On 


CLINICAL  AND   PATHOLOGICAL  STUDIES  197 

cut  section  these  areas  are  sharply  circumscribed,  pure  j-ellow  in  color,  and  extend 
for  a  depth  of  1  mm.  into  the  liver  substance.  Marked  by  scissors  for  identification. 
On  cut  section  there  are  several  other  of  these  yellowish  areas.  One  found  in  the 
depth  of  the  liver  protruding  slightly  from  the  cut  surface. 

On  cut  section  of  the  liver  the  surface  is  distinctly  uneven,  and  presents  a  slightly 
nutmeg  appearance.  The  uneven  appearance  is  caused  by  small  circumscribed 
raised  areas,  sometimes  confluent,  from  pin-point  to  pin-head  in  size,  which  are  of 
a  bro\\'n  color,  and  which  are  situated  upon  a  grayish  background.  These  small 
islands  of  tissue  are  surrounded  by  a  narrow  red  zone.  In  the  depth  of  the  liver 
another  of  these  j'ellowish  areas  is  found,  measuring  10  X  6  mm. 

Stomach  is  filled  vnih  reddish  black  material.  The  omentum  contains  nu- 
merous small  hemorrhages.  The  mucous  membrane  of  the  stomach  is  covered  with 
blackish  mucoid  material,  on  removal  of  which  the  mucosa  appears  grayish  yellow 
with  small  dark  red  hnes  and  points  due  to  injection  of  the  vessels.  Mucous 
membrane  of  the  stomach  is  shghtly  swollen,  but  otherwise  appears  normal.  The 
surface  of  the  intestine  is  bile-stained  as  alreadj^  noted.  The  mesocolic  glands  are 
more  markedly  reddened  than  the  mesenteric  glands  situated  at  a  distance.  On 
opening  the  lower  portion  of  the  large  intestine  it  is  found  to  be  practically  normal 
and  to  contain  normal  faeces.  Several  feet  above  the  rectum  there  are  several 
small  hemorrhages  about  the  peritoneal  vessels.  Further  up  the  large  intestine 
is  filled  with  a  reddish  brown  material  which  has  a  very  foul  odor.  This  condition 
extends  to  the  large  intestine  as  far  up  as  the  cecum.  On  scraping  off  this  reddish 
browTi  material  the  mucous  membrane  appears  practically  normal.  In  the  neigh- 
borhood of  the  cecum,  however,  the  mucous  membrane  is  profusely  reddened 
throughout.  This  process  does  not  extend  beyond  the  valve,  the  mucous  mem- 
brane of  the  ileum  appearing  normal  as  well  as  its  contents.  The  solitary  follicles 
are  distinctly  \'isible,  but  not  swollen.  The  Peyer's  patches  are  also  not  swollen. 
In  the  jejunum  there  are  evidences  of  altered  blood.  The  pancreas,  bladder  and 
genital  organs  appear  normal.  The  other  organs  also  appear  normal.  There  are 
some  erosions  at  the  edges  of  the  tongue,  and  slight  injection  of  the  mucous  vessels 
of  the  trachea. 

Bacteriology.  Cultures  were  made  from  the  heart,  spleen,  liver  and  kidneys 
and  also  microscopical  preparations. 

Histological  Examination 

Heart.  The  muscle  fibers  show  one  or  more  vacuoles  situated  in  the  central 
portion  suggestive  of  fat. 

Lung.  Areas  of  hemorrhage.  In  one  such  area  there  is  a  mass  of  dense  cellular 
exudate  filling  the  bronchus,  and  the  alveoli  are  filled  with  polymorphonuclear 
leukocytes  and  large  endothelial  cells  as  well  as  red  blood-corpuscles. 

In  another  section  some  of  the  similar  vessels  show  thrombi ;  the  appearances 
being  those  seen  in  infarctions. 

Stomach.    Presents  no  lesion. 

Large  Intestine.     Appears  normal. 

Liver.  The  larger  portion  of  the  liver  parenchyma  is  destroyed.  The  remaining 
liver  cells  are  grouped  chiefly  around  the  portal  canals  and  to  a  lesser  extent  around 
the  central  veins.  Towards  the  periphery  of  the  normal  areas  the  cells  are  more 
markedly  vacuolated  than  elsewhere,  and  many  of  the  degenerating  cells  stain  red 
and  appear  .somewhat  granular.  Distributed  throughout  the  degenerated  and  ne- 
crotic portions  there  is  a  considerable  amount  of  pigment  situated  to  a  large  extent 
within  the  phagocytic  cells  and  suggesting  complication  with  malaria.    Many  of 


198  YELLOW  FEVER 

the  vacuolated  liver  cells  are  distended  to  huge  proportions,  and  mitotic  figures  are 
present.  There  is  a  moderate  amount  of  pigment  situated  within  the  bile  capil- 
laries. The  blood  is  distributed  throughout  the  necrotic  areas,  and  it  is  not  always 
confined  in  well  defined  blood-vessels.  There  are  syncytial  like  masses  in  the  region 
of  the  portal  canals,  possibly  an  attempt  at  regeneration  on  the  part  of  the  bile 
ducts,  although  they  have  much  the  appearance  of  giant-cells  in  certain  places. 
There  are  also  numerous  foci  infiltrated  with  lymphoid  and  plasma-cells,  somewhat 
irregular  in  distribution,  but  more  frequently  near  the  portal  canal. 

Pancreas.  There  is  no  inflammatory  or  destructive  change  observed.  The 
glands  throughout  are  devoid  of  zymogen  granules.  Rounded  globules  occur 
within  the  glandular  epithelium  in  certain  areas. 

Adrenal.  Parenchyma  intact  and  cells  non- vacuolated.  There  appears  to  be 
some  oedema  of  the  interstitial  tissue. 

Kidney.  Glomeruh  slightly  injected.  Epithelium  of  the  convoluted  tubules 
shows  granular  and  somewhat  vacuolated  cytoplasm.  There  is  an  occasional  hyaUne 
cast.     In  the  medulla  there  are  several  minute  foci  of  hemorrhage. 

Bladder.     Presents  no  lesion. 

Spleen.  There  is  considerable  golden-brown  and  black  pigment  situated  within 
phagocytic  cells  characteristic  of  malaria.  The  lymph-folhcles  show  regularly  a 
lesion  characterized  by  an  accumulation  of  fibrinoid  material  in  the  form  of  a  coarse 
reticulum  and  including  in  its  meshes  various  types  of  cells. 

Lymph-node.  Shows  large  amount  of  carbon  pigment.  No  marked  pathological 
process. 

Anatomical  Diagnosis.  Infarction  of  lung.  Chronic  pleuritis.  Extensive  degen- 
eration and  necrosis  of  hver.  Pigmentation,  probably  malarial.  Acute  splenitis 
with  fibrinoid  changes  in  follicles.     Cloudy  swelling  of  kidney. 

It  is  not  deemed  necessary  to  give  the  notes  of  the  other 
cases  of  yellow  fever  from  which  we  made  examinations  of  the 
blood.  From  the  histories  given  above,  it  is  evident  that  the 
diagnosis  of  yellow  fever  was  unquestionable.  In  eleven  of 
the  most  severe  cases  of  yellow  fever  observed  by  us  in  which 
the  cases  referred  to  above  are  included,  numerous  inoculations 
of  the  blood  were  made  into  the  ordinary  laboratory  media 
such  as  agar,  blood-serum,  and  egg  media,  and  with  the  various 
methods  described  by  Noguchi  from  the  Rockefeller  Institute 
for  the  cultivation  of  spirochaetae.  Both  aerobic  and  anaero- 
bic preparations  of  ascitic  fluid  alone,  and  of  ascitic  fluid  with 
agar,  or  in  other  instances  with  fragments  of  the  rabbit's  testis, 
were  employed.  In  yellow  fever  cases  Nos.  8,  20,  21,  and  26, 
inoculations  were  also  made  in  these  media  from  the  heart's 
blood  and  the  solid  visceral  organs  at  necropsy.  In  none 
of  the  cultures  was  any  organism  found  which  appeared  to 
bear  any  etiological  relationship  to  yellow  fever.  In  Case  21, 
streptococci  were  isolated  from  the  lungs  and  heart's  blood. 


BLOOD   EXAMINATIONS  199 

In  the  study  of  numerous  fresh  and  stained  blood  prepara- 
tions from  the  yellow  fever  cases  which  were  observed,  we  did 
not  discover  bodies  which  we  regarded  as  of  a  parasitic  nature, 
and  at  the  same  time  the  cause  of  the  disease  yellow  fever. 
In  Cases  Nos.  22  and  23,  young  forms  of  the  tropical  malarial 
parasite  were  found  in  the  red  blood-corpuscles.  Giemsa's 
and  \Yright's  stains  were  both  employed,  after  fixation  usually 
with  absolute  methyl  alcohol  and  in  some  instances  with  ethyl 
alcohol. 

During  the  past  year  Alacfie  and  Johnston  ^  have  reported 
finding  bodies  in  the  red  blood-corpuscles  similar  in  appear- 
ance to  those  described  by  Seidelin.  They  found  these 
elements  in  practically  every  case  of  yellow  fever  examined, 
and  also  in  the  blood  of  guinea  pigs,  dogs,  and  rats  that  had 
been  inoculated  with  blood  from  human  cases.  In  guinea 
pigs  these  bodies  were  particularly  common. 

Fajardo  -  has  also  very  recently  reported  the  presence  of 
these  bodies  in  the  blood  of  one  case  of  yellow  fever.  Con- 
siderable controversy  has  taken  place  regarding  the  nature  of 
these  bodies  and  it  appears  to  be  the  consensus  of  opinion 
today  that  they  are  not  of  a  parasitic  nature.  Agramonte  ^ 
has  pointed  out  that  they  have  been  found  in  many  conditions 
other  than  in  yeUow  fever,  and  that  the  bodies  described  by 
Seidelin  would  not  be  able  to  pass  through  a  Berkefeld  filter, 
while  the  virus  of  yellow  fever  certainly  does.  He  also  draws 
attention  to  the  fact  that  Seidelin  has  found  these  bodies  in 
many  cases  which  have  presented  no  signs  of  yellow  fever. 

Guiteras  ^  has  recently  objected  to  the  diagnosis  of  a  case 
of  yellow  fever  in  Jamaica  in  which  case  the  diagnosis  was 
made  by  Seidelin  largel}^  apparently  by  the  finding  of  these 
supposedly  parasitic  bodies.  Guiteras  points  out  that  the 
disease  is  not  supposed  to  exist  in  Jamaica,  and  that  various 
clinical  features  make  it  very  improbable  that  the  case  was 

'  Macfie  and  Johnston:  Proc.  Roy.  Soc.  Med.,  1914,  Jan.,  vii,  No.  3,  Medical  Section, 
p.  53. 

Yellow  Fever  Bureau  Bull.,  Liverpool,  1914,  iii,  121. 
2  Fajardo:  Revi.sta  Med.  de  Yucatan,  1914,  Jan.,  ix.  No.  3,  p.  53. 
»  .A^rarnonte:   Proc.  xviith  Inter.  Con^-  Med.,  Lond.,  1913,  Sect.  21,  Part  II,  p.  77. 
*  (iuiteras:   Yellow  Fever  Bureau  Bull.,  Liverpool,  1914,  iii,  110. 


200  YELLOW  FEVER 

one  of  this  disease.  Particularly  striking  in  this  respect  was 
the  absence  of  fever,  the  pulse  rate,  and  lack  of  intense  jaun- 
dice. Agramonte  very  justly  points  out  that  in  connection 
with  the  possible  inoculations  of  these  bodies  into  guinea  pigs 
he  has  seen  similar  bodies  in  guinea  pigs  treated  with  normal 
human  blood  and  in  various  human  conditions.  Schilling- 
Torgau  ^  also  does  not  think  that  these  bodies  are  parasites 
but  that  they  represent  structures  occasionally  present  within 
the  erythrocytes  of  man  and  animals.  Wenyon  ^  in  writing  of 
these  bodies  described  by  Seidelin  says  even  if  one  accepts  the 
statement  that  the  bodies  are  present  in  cases  of  yellow  fever 
and  absent  in  other  conditions  the  author  has  yet  to  prove 
that  they  are  parasitic  and  if  so  are  the  etiological  factor  in 
the  production  of  yellow  fever.  Wenyon  and  Low^  also 
believe  they  have  found  the  same  bodies  in  normal  guinea 
pigs'  red  blood-corpuscles. 

1  Schilling-Torgau:  Beiheft  1,  Archiv.  f.  Schiffs- u.  Tropenhyg.,  Leipz.,  1912,  xvi,  87. 

Archiv.  f.  Schiffs-  u.  Tropenhyg.,  Leipz.,  1912,  xvi,  373. 

2  Wenyon:  Tropical  Diseases  Bull.,  Lond.,  1912-1913,  I,  249. 

3  Wenyon  and  Low:  J.  Trop.  Med.  and  Hyg.,  Lond.,  1914,  xvii,  No.  24,  369. 


IX 

LINGUATULIDA 

Crocodiles  are  numerous  in  the  rivers  near  Guayaquil,  and  a 
number  of  them  were  obtained  from  the  Guayas  river  varying 
from  about  four  to  ten  and  one-half  feet  in  length.  (Plate 
XLVii.)  The  specimens  obtained  proved  to  be  of  the  same 
species,  and  one  preserved  and  brought  to  this  city  has  been 
identified  by  Dr.  Thomas  Barbour,  Curator  of  Oceanica  and 
instructor  upon  Poisonous  Reptiles  in  this  School,  as  Crocodilus 
americanus  (Laurenti) .  According  to  Barbour  this  species  has 
a  wide  range  of  distribution,  having  been  found  in  south  Flor- 
ida, the  Greater  Antilles,  central  and  northern  South  America. 
It  reaches  the  southern  limit  of  its  distribution  on  the  west 
coast  of  South  America  in  the  Guayas  river  area.  In  three  of 
four  of  the  crocodiles  examined  a  linguatulida  of  the  genus 
Porocephalus  was  found  in  the  lungs.  (Plate  xlviti.)  In  one 
instance  the  lung  showed  at  the  apex  a  considerable  abnormal 
fibrosis.  A  nodular  mass  was  found  measuring  about  five  mil- 
limeters in  diameter,  which  on  section  consisted  of  dry  gray- 
ish cheesy  material.  On  cutting  in  various  directions  through 
the  lung,  elongated  and  slightly  flat  parasites  were  found  vary- 
ing from  0.5  to  2.5  centimeters  in  length  usually,  having 
in  general  a  pale  pink  color,  with  bright  red  and  dark  brown 
areas  visible  in  their  interior.  Some  of  these  were  encysted  or 
situated  in  the  lung  substance  proper,  and  none  were  found 
free  in  the  bronchi  or  in  the  vessels.  The  lungs  of  two  other 
crocodiles  were  placed  over  night  in  formalin.  The  following 
morning  decomposition  had  already  commenced.  Upon  dis- 
section of  all  of  the  lungs  a  large  number  of  linguatulidae  were 
found.  Some  of  these  were  free  in  the  bronchi  and  in  the  large 
air  chambers.  Possibly  some  of  them  had  migrated  after  the 
death  of  the  host.  A  number  of  the  ])arasites  were  somewhat 
macerated  and  had  lost  their  pink  color.    The  parasites  were 


202  LINGUATULIDA 

referred  to  William  M.  Wheeler,  Professor  of  Economic  Ento- 
mology in  this  University  and  in  charge  of  the  instruction  in 
Tropical  Entomology  in  this  School,  who  reports  that  while 
the  species  is  very  closely  related  to  Diesing's  Porocephalus 
gracilis,  it  is  evidently  a  new  and  undescribed  one.  Professor 
Wheeler's  account  of  this  parasite  is  found  in  the  Appendix 
on  page  207;  to  it  he  has  given  the  name  of  Porocephalus 
crocodili. 


Fig.  1.  —  Crocodile  (Crocodilus  americanus)  shot  in  the 
GuAYAS  River. 


Fig.  2.  —  Daule  River.     Favorite  hiuint  of  crocodiles. 
Plate  XLVII.  —  Ecuador. 


Fig.  2.  —  Cross-section  of  Adult  (Porocephalus 
crocodili  sp.  nov.).      X  114. 


Fig.  1.  —  Adult  Female  of 
LiNGUATULiDA  {Porocephaht.s 
crocodili  sp.  nov.).  AboutX21. 


"^^t 


Fig.  3.  —  Gheateu  Magnification  of  Head. 
Showing  mouth  and  liooklcts.     About   X  61. 


Plate  XLVIII.  —  Linguatulida  from  lun(;  of  ckocouile 


X 

ACKNOWLEDGMENTS 

In  connection  with  the  investigations  outlined  in  this  report  we  wish  to  ex- 
press publicly  our  thanks,  particularly  to  the  Corporation  of  Harvard  Univer- 
sity for  generously  providing  funds  for  the  maintenance  of  the  expedition 
and  to  the  Dean  of  the  Harvard  Medical  School  and  the  Dean  of  the  Gradu- 
ate School  of  Medicine  for  their  interest  and  assistance  in  the  organization 
of  the  expedition;  to  the  United  Fruit  Company  of  Boston  for  generously 
furnishing  us  with  transportation  from  New  York  to  Colon  and  return; 
to  the  Royal  Mail  Steamship  Company  and  the  Pacific  Steamship  Naviga- 
tion Company  for  furnishing  us  free  transportation  from  Balboa  to  Guaya- 
quil and  from  Callao  to  Balboa,  and  to  the  Panama  Railroad  Company  for 
courtesy  in  regard  to  handling  our  baggage  across  the  Isthmus.     We  also 
wish  to  express  particularly  our  gratitude  to  Mr.  J.  W.  Blaisdell,  assistant 
manager  of  the  Central  Railroad  Company  of  Peru,  who  very  generously 
on  several  occasions  furnished  us  with  special  railway  facilities  in  our 
studies  carried  on  between  Lima  and  Oroya,  and  to  Dr.  W.  F.  Bailey,  Cerro 
de  Pasco  Railway,  for  many  kindnesses  during  our  expedition  from  Oroya 
to  Cerro  de  Pasco.     To  the  following  physicians  and  officials  we  wish  to 
express  our  grateful  acknowledgment  for  many  kindnesses  and  for  assisting 
us  in  obtaining  material  in  connection  with  our  studies:  Drs.  Deeks,  Perry, 
Caldwell,  Darling  and  James  in  Panama;    Dr.  Parker,  Consul  General 
Baker  and  Dr.  Pareja  and  other  officials  of  the  Board  of  Health  in  Guaya- 
quil; President  Billinghurst  of  Peru,  the  American  minister,  Mr.  Henry- 
Clay   Howard,   the  introductor   of   ministers,    Mr.    Cisneros,    and   Drs. 
Odriozola,  Arce,  Grana,  Barton,  Hercelles  and  Matto  in  Lima.     Dr.  Pareja 
particularly  assisted  us  in  obtaining  pathological  material  in  relation  to 
yellow  fever  and  Drs.  Odriozola,  Arce,  Graiia  and  Barton  in  finding  cases 
of  verruga  peruviana.     Dr.  Hercelles  supplied  us  with  a  number  of  tissues 
from  Oroya  fever  cases.     We  also  wish  to  express  our  great  appreciation 
of  the  very  valuable  assistance  rendered  us  by  Dr.  J.  C.  Tello,  Director  of 
the  Ethnologic  Museum,  Lima,  in  facilitating  our  obtaining  material  and  in 
assisting  us  in  making  arrangements  in  regard  to  some  of  our  investigations. 
Without  Dr.  Tello's  assistance  some  of  our  investigations  would  probably 
not  have  been  brought  to  as  successful  an  issue  as  they  were.     Finally,  we 
wish  to  express  our  deep  appreciation  of  the  efficient  services  rendered  by 
Miss  Nora  Dwyer,  who  accompanied  us  as  secretary  on  the  expedition. 

The  drawings  of  the  microscopical  preparations  accompanying  the  re- 
port were  made  by  Miss  E.  R.  Piotti,  and  the  photomicrographs  by  Mr. 
L.  Brown.  Miss  C.  M.  Casassa  has  been  of  much  assistance  in  reading  the 
proof,  and  we  are  greatly  indebted  to  Mr.  C.  C.  Lane  for  having  seen  the 
report  through  the  Harvard  University  Press. 

203 


APPENDIX 


APPENDIX 

I.    A   NEW   LIXGUATULID   FROM   ECUADOR 
By  William  Morton  Wheeler 

Professor  of  Economic  Entomology 

Amoxg  the  forms  described  Ijy  Diesing  in  his  well-known  monograph  of 
the  Linguatulids  ^  are  two  South  American  species  called  respectively 
Pentastoma  gradle  and  P.  furcocercum,  which  differ  from  the  other  species 
in  ha\ing  the  hamuli,  or  hooks  surrounding  the  mouth  geminate,  or  double. 
These  species  were  later  transferred  to  the  genus  Porocephalus  Humboldt 
when  this  name  was  substituted  for  Pentastoma  Rudolphi  and  restricted  to 
species  having  a  cylindrical  body,  without  lateral  diverticula  of  the  body- 
cavity  in  each  annulus.  The  two  species  described  by  Diesing,  however, 
should  probably  constitute  a  distinct  genus,  -with  P.  gracile  Diesing  as  the 
type.  A  third  undescribed  species  belonging  to  this  same  group  and 
recently  taken  by  the  Harvard  Expedition  to  South  America  from  the 
lungs  of  a  crocodile  {Crocodilus  americanus)  captured  in  the  Guayas 
river,  at  Guayaquil,  Ecuador,  were  referred  to  me  for  identification.  The 
specimens  were  mostly  small  and  immature,  but  they  included  an  adult 
male  and  female  and  were  sufficiently  well  preserved  to  enable  me  to  draw 
up  the  following  specific  diagnosis : 

Porocephalus  crocodili  sp.  no  v. 

The  specimens  vary  from  3-25  mm.  in  length.  The  body  is  pale  yellow, 
slender,  cylindrical,  blunt  at  the  ends,  usually  slightly  broader  anteriorly, 
with  the  posterior  half  or  third  curved  ventrally.  The  annuli  are  numer- 
ous, 105  in  one  male  specimen  measuring  25  mm.,  and  75  in  a  specimen 
measuring  only  5  mm.  They  are  very  narrow,  but  quite  sharply  marked 
off  from  one  another  both  in  large  and  small  specimens,  except  at  the  ex- 
treme posterior  end.  Anteriorly  the  head  bears  a  pair  of  small  rounded 
sensory  papillae  and  a  second  pair  of  similar  but  less  prominent  structures 
further  apart  on  its  dorsolateral  surface.  The  pores  on  the  borders  of  the 
annular  folds  are  very  small,  and  rather  indistinct.  The  mouth  opening  is 
large  and  elHptical  or  more  rarely  circular,  as  shown  in  Plate  xlviii,  Figs. 
1  and  3.  It  is  surrounded  by  four  pairs  of  double  chitinous  hooks  which 
arise  from  elongated  slits  with  welt-like  margins.  In  each  pair  the  pos- 
terior hook  is  strongly  curved,  the  anterior  distinctly  more  attenuate  and 

'  Versuch  einer  Monographie  der  Gattung  Pentastoma,  Ann.  Wien.  Mus.  Naturgesch. 
I,  1836,  pp.  1-32,  pl.s.  1-4. 

207 


208  APPENDIX 

more  nearly  straight.  In  the  male  the  genital  papilla,  which  is  situated 
in  the  mid- ventral  line  on  the  second  annulus  behind  the  mouth,  is  small 
but  projecting. 

The  specimens  when  found  were  partly  free  in  the  bronchial  cavities 
and  partly  encapsuled  in  the  lung  tissues  of  the  crocodile.  They  were  pre- 
served in  formalin. 

P.  crocodili  is  evidently  very  closely  related  to  Diesing's  P.  gracilis,  but 
this  author's  figures  show  both  hooks  of  a  pair  to  be  equally  developed  and 
strongly  curved,  which  is  certainly  not  the  case  in  P.  crocodili.  In  the  latter, 
moreover,  the  hooks  are  much  smaller,  less  projecting  and  less  heavily 
chitinized.  Diesing  based  his  species  on  a  large  number  of  specimens  col- 
lected by  Natterer  at  Cuyaba,  Caicara  and  Villa  Maria,  Brazil.  They 
were  found  either  free  in  the  body  cavity  or  encapsuled  in  the  viscera  and 
mesenteries  of  lizards,  snakes  and  fish.  Among  the  lizards  cited  as  hosts 
are  two  species  of  Podinema,  among  the  snakes,  species  of  Elaps,  Coluber, 
Bothrops,  Pseuderys,  Tropidonotus  and  Eunectes,  and  among  the  fish,  species 
of  Lohotes,  Silurus,  Piara,  Pimelodes,  Salmo,  Serrosalmo,  Clupea,  Erythrinus, 
Symbranchus,  Gymnotus,  Sternarchus  and  Raja.  The  other  species  of  Poro- 
cephalus  with  double  hooks  (P.  furcocercum)  was  also  taken  by  Natterer  at 
Cuyaba,  in  the  body  cavity,  lungs  and  mesentery  of  snakes  {Coluber  and 
Spilotes)  and  Amphisbcena  flavescens.  This  Linguatulid  is  readily  distin- 
guished from  gracilis  and  crocodili  by  the  peculiarly  bifurcate  posterior  end 
of  the  body  and  the  three  papillae  around  the  base  of  each  pair  of  hooks. 
In  this  case  the  hooks  are  more  like  those  of  P.  crocodili,  as  Diesing  says: 
"  Der  obere  ist  fast  gerade  und  kiirzer,  der  untere  langer,  mehr  nach  innen 
gebogen."  Had  this  been  the  case  in  gracilis  Diesing  could  hardly  have 
failed  to  call  attention  to  the  difference  in  his  description  or  to  show  it  in 
his  figures. 


APPENDIX  209 

II.    SOIME  FLIES   OF  THE  FAMILY  PHORID.E  OBTAINED  BY  THE 

EXPEDITION,   WITH  NOTES   ON  A   SPECIES  POSSIBLY 

ASSOCIATED   WITH  EXTERNAL   MYIASIS   IN   :MAN 

By  Charles  T.  Brues 

Assistant  Professor  of  Economic  Entomology 

Aphioch^ta  ferruginea  Brunetti 

While  in  Buenaventura,  Colombia,  on  May  14,  some  scrapings  were 
taken  from  the  skin  of  a  negro  affected  ^\dth  caraate,  and  placed  in  two  test- 
tubes  containing  an  agar  medium.  These  were  well  plugged  with  cotton, 
fastened  in  place  with  paraffin  and  placed  in  a  wire  basket  together  with 
a  number  of  similar  tubes  inoculated  from  other  sources.  On  examining 
the  tubes  after  reaching  Guayaquil  on  May  18,  a  number  of  minute  Dipter- 
ous larvae  were  found  feeding  on  the  surface  of  the  agar  in  the  two  tubes 
mentioned,  although  there  were  none  present  in  any  of  the  other  tubes 
which  had  been  kept  in  the  same  basket.  From  this  it  appeared  that  the 
larvae  must  have  been  derived  from  the  skin  of  the  negro  rather  than  have 
worked  into  the  tube  later,  as  all  the  tubes  contained  the  same  agar  medium. 
The  larvae  developed  very  slowly,  but  finally  pupated  during  the  first  week 
of  June  and  began  to  emerge  as  adult  flies  between  June  26  and  July  1. 
They  then  proved  to  be  a  species  of  Phoridae  belonging  to  the  genus  Aphio- 
chaeta. 

Although  the  evidence  is  not  complete,  that  either  eggs  or  freshly 
hatched  larvae  were  present  on  the  skin  of  the  negro  when  the  scrapings 
were  made,  it  seems  very  probable  that  such  was  the  case.  Even  so,  it  is 
of  course  doubtful  if  this  is  a  regular  habit  of  this  species.  However,  the 
material  was  obtained  from  the  back  of  the  patient  and  the  skin  at  this 
point  was  exceedingly  rough  and  scaly,  so  that  eggs  or  larvae  of  such  min- 
ute size  would  readily  have  escaped  notice  at  the  time  the  tube  was  inocu- 
lated. 

There  is  some  doubt  concerning  the  identity  of  this  species  and  on  this 
account  there  is  given  a  description  based  on  the  specimens  reared  from  the 
larvae  obtained  at  Buenaventura.  Aphiochceta  ferruginea  was  first  described 
by  Brunetti  from  India  (Rec.  Indian  Museum,  vol.  7,  p.  84  (1912)),  but  has 
been  recorded  by  Austen  from  West  Africa,  the  West  Indies  and  Central 
America.  Cotypes  of  Brunetti's  species  have  been  examined  by  the  writer 
and  also  what  appears  to  be  the  same  species  from  Abyssinia.  There  is 
considerable  variation  however  in  the  several  lots  and  more  than  one  species 
may  be  concerned. 

The  Indian  A.  ferruginea  has  been  found  to  cause  myiasis  of  the  intes- 
tines and  was  long  ago  reported  on  by  Baker  in  the  Proceedings  of  the 
Burma  Branch  of  the  British  Medical  Association  for  1891,  pp.  U-IG  and 
28-29.    In  British  Honduras,  Heuser  has  bred  the  Central  American  form 


210  APPENDIX 

from  larvae  extracted  from  an  Indian's  foot  and  says  that  the  species  is 
abundant  about  the  mangrove  swamps  at  Belize  where  it  occurs  about  de- 
composing shell-fish.^  The  specimens  from  Abyssinia  were  reared  from 
dead  and  imperfectly  dried  beetles,  and  Brunetti,  Smith  and  Austen  have 
reared  adults  from  decaying  insects  and  putrid  meat. 

Aphioch^ta  ferruginea  Brunetti  (Plate  xxxiii,  fig.  3) 

(J  9  Length  1.3-1.6  mm.  Brownish-yellow;  front  fuscous,  black  about  the  ocelli; 
thorax  above  brownish;  abdomen  above  piceous  basally  and  black  apically,  the  base 
of  the  second  segment  and  a  median  spot  on  the  third  and  fourth  segments  orange-yellow; 
hind  femora  tipped  with  black ;  pleurae  and  venter  basally,  pale  testaceous.  Wings  hya- 
line, venation  pale  fuscous.  Front  about  as  broad  as  high,  with  four  proclinate  bristles 
of  nearly  equal  size,  the  lower  pair  half  as  far  apart  as  the  upper  pair,  the  latter  in  a  line 
with  the  lower  reclinate  bristle  of  the  next  row  and  slightly  closer  to  it  than  to  the  me- 
dian line.  Lateral  reclinate  bristle  of  this  row  high  up  and  but  little  nearer  the  eye 
than  the  median  bristle.  Four  reclinate  bristles  of  next  row  above  equidistant,  forming 
a  nearly  straight  line;  bristles  of  oceUar  row  normal,  no  larger  than  the  others.  Ocellar 
tubercle  and  median  frontal  groove  present;  surface  of  front  subshining.  Cheeks  each 
with  two  equal,  downwardly-directed  macrochsetse.  Antennae  small,  rounded,  fulvous, 
with  a  densely  pubescent  arista  that  is  distinctly  longer  than  the  head-height.  Palpi 
pale  yellow,  moderately  large,  with  five  or  six  large  macrochaetae  on  apical  third  below, 
bare  basally.  Scutellum  with  four  bristles,  the  lateral  pair  scarcely  smaller  than  the 
others;  mesonotum  with  one  pair  of  widely  separated  dorsocentral  macrochaetae,  but 
without  other  bristles  along  its  posterior  margin.  Mesopleura  bare.  Abdomen  with  a 
small  tuft  of  bristly  hairs  at  each  side  of  the  second  segment,  and  with  a  number  of 
longer  conspicuous  bristles  on  the  apex  of  the  last  two  segments,  especially  in  the  female. 
Hypopygium  of  male  small,  the  anal  protuberance  pale  yellow.  Fore  legs  distinctly  thick- 
ened, especially  the  tibiae  which  are  densely  clothed  with  stouter  bristles  than  usual  and 
show  a  distinct  row  of  about  ten  setulae  on  the  postero-dorsal  surface;  middle  tibia  with 
a  single  row  of  sparsely  placed  setulae  on  the  postero-dorsal  surface;  hind  tibia  with  a 
similarly  placed  series  of  8-10  moderately  large  setulae.  Costal  vein  reaching  the  middle 
of  the  wing,  its  cilia  rather  long  and  closely  placed;  first  vein  entering  costa  midway 
between  the  humeral  cross- vein  and  tip  of  third  vein;  second  vein  entering  costa  three 
times  as  far  from  tip  of  first  as  from  tip  of  third,  the  cell  at  the  furcation  nearly  twice 
as  high  as  long;  fourth  vein  evenly,  but  not  strongly  curved  for  its  entire  length;  fifth 
and  sixth  slightly  bisinuate;  seventh  nearly  straight,  distinct.  Hal  teres  pale  brownish, 
strongly  darkened  on  the  apical  half  of  the  knob. 

Among  the  other  Phoridae  obtained,  one  species  is  new  to  science  and  is 
described  below: 

Aphioch^ta  incarum  sp.  nov. 

^  Length  1.3  mm.  Thorax  pale  fuscous,  front  black,  antennae  deep  yellow  with 
apical  half  of  third  joint  blackened,  palpi  pale  yellow,  pleurae  and  legs  testaceous.  Ab- 
domen above  piceous,  brownish  on  the  fifth  and  sixth  segments,  hypopygial  appendage 
pale  yellow,  halteres  pale  yellow.  Wings  hyaline.  Front  very  slightly  wider  than  high; 
only  two  supra-antennal  bristles,  each  strong  and  half  as  long  as  the  height  of  the  front. 
Bristles  of  lowest  reclinate  row  forming  a  closely  placed  pair  at  each  lower  angle  of  the 
front,  very  close  to  the  eye;  bristles  of  next  row  forming  a  line  bowed  down  medially, 

1  Trans.  Soc.  Trop.  Med.  &  Hyg.,  Lond.,  1910,  iii,  230. 


APPENDIX  211 

its  lateral  bristles  very  close  to  the  eye,  its  medial  ones  closer  to  one  another  than  to  the 
lateral  bristles.  Ocellar  tubercle  and  median  frontal  line  present;  surface  of  front  sub- 
shining.  Antennae  rather  large,  broadly  oval,  the  black  on  the  third  joint  sharply 
marked.  Palpi  with  short,  weak  bristles.  Post-ocular  cilia  moderate,  cheeks  each  with 
a  stout  macrochaeta  and  a  series  of  four  small  bristles  running  upwards  toward  the  an- 
tenna. Mesonotum  shining,  finely  hairy,  with  a  single  pair  of  dorsocentral  macrochaetae 
placed  on  the  posterior  edge  of  the  mesonotum  opposite  the  lateral  corners  of  the  scu- 
tellum.  Scutellum  with  two  large  bristles  in  addition  to  a  lateral  microscopic  hair.  Me- 
sopleura  bare,  without  bristles  or  hairs  above.  Abdomen  with  none  of  its  segments 
noticeably  lengthened  or  shortened,  along  the  sides  with  a  few  conspicuous  short  bristles, 
two  or  three  to  each  segment;  on  the  sixth  and  seventh  segment  these  are  longer  and 
form  a  series  along  the  posterior  margin  also.  Abdomen  opaque,  except  the  seventh  seg- 
ment which  is  shining  and  jet-black.  Hypopygium  shining  black.  Hind  tibiae  with  dis- 
tinct, but  not  strong  setulae  placed  in  a  single  line  on  the  postero-dorsal  surface.  Wings 
long  and  narrow,  the  costal  vein  just  attaining  the  middle,  its  bristles  long  and  sparse, 
only  four  between  the  tips  of  the  first  and  third  veins;  first  vein  ending  twice  as  far 
from  the  humeral  cross-vein  as  from  the  tip  of  the  third;  second  vein  ascending  steeply 
to  the  costa,  forming  a  cell  as  long  as  its  own  length,  entering  the  costa  one-half  nearer 
to  the  tip  of  the  third  vein  than  to  the  tip  of  the  first;  fourth  vein  slightly  curved,  re- 
curved strongly  at  tip;  seventh  distinct;   all  veins  rather  pale  and  weak. 

One  male.    Lima,  Peru,  June,  1913. 

This  species  is  related  to  A.  evarthce  Malloch^  from  the  United  States, 
but  differs  in  the  absence  of  the  two  lower  proclinate  bristles  on  the  front 
and  the  black,  instead  of  yellow  anal  protuberance.  It  does  not  resemble 
closely  any  described  South  American  species. 

1  Proc.  U.  S.  Nat.  Mus.,  1912,  xHii,  472. 


212  APPENDIX 

III.    NOTES  ON  PERUVIAN  MOSQUITOES  AND  MOSQUITO 

LITERATURE 

By  Feederick  Knab 

Bureau  of  Entomology,  Department  of  Agriculture, 
on,  D.  C. 


In  a  recent  paper  Dr.  H.  G.  Dyar  and  the  writer  have  indicated  the 
mosquitoes  so  far  known  from  the  Andean  and  coast  regions  of  Peru.^ 
Newstead  and  Thomas  have  reported  a  limited  number  of  species  from 
Iquitos,  in  the  forest  region  of  eastern  Peru.^  Professor  C.  T.  Brues  has 
submitted  for  my  examination  a  paper  containing  descriptions  of  two  sup- 
posedly new  species  of  Peruvian  mosquitoes,  published  at  Lima  in  1907. 
The  paper  in  question  is  rare  and  has  until  now  escaped  the  notice  of  sys- 
tematists.  Its  authors  are  M.  0.  Tamayo  and  C.  A.  Garcia  and  it  deals 
with  Lake  Huacachina  and  other  smaller  nearby  lakes  which  are  reputed 
to  have  therapeutic  qualities.^  A  part  of  the  report  is  taken  up  with  the 
natural  history  of  the  lake,  and  in  that  connection  two  species  of  mosquitoes 
are  figured  and  described  as  new.  On  page  xxxv  of  the  extract  "  Ano- 
pheles peruvianus  Tamayo  "  is  proposed,  and  on  page  xxxvii  "  Culex 
raymondii  Tamayo."  The  descriptions,  although  lengthy  are  unsatis- 
factory. Large,  but  rude,  figures,  on  five  plates,  illustrate  both  sexes  of 
the  imagos,  the  larvae  and  pupae  of  the  two  species. 

The  writer  is  of  the  opinion  that  both  these  proposed  new  species  are 
synonyms  of  well  known,  widely  distributed  species.  This  opinion  is  sup- 
ported by  specimens  from  Peru. 

Anopheles  peruvianus  is  without  doubt  identical  with  A.  pseudopuncti- 
pennis  Theobald  in  spite  of  discrepancies  in  the  description  and  still  more 
in  the  figures.  From  the  description  and  figure  of  the  female  the  following 
essential  characters  may  be  abstracted :  the  head  has  on  the  vertex  a  patch 
of  white  twisted  scales  which  project  in  a  tuft  between  the  eyes.  The 
palpi  are  black  scaled,  with  narrow  pale  rings  at  the  articulations  and  the 
last  joint  yellowish  white.  Thorax  with  the  median  section  whitish  (gray) 
and  with  three  characteristic  lines.  Legs  dark,  with  narrow  pale  rings  at 
the  bases  of  the  tarsal  joints,  which  are  entirely  covered  with  very  dark 
scales.  Wings  with  the  costa  black  on  the  basal  two-thirds,  followed  by  a 
yellowish  white  spot  which  originates  at  the  tip  of  the  subcostal  vein;  this 
is  again  followed  by  black,  beyond  which  the  extreme  tip  is  again  whitish 
scaled.     The  subcostal  and  first  veins  show  three  black  spots,  the  first  of 

1  New  mosquitoes  from  Peru.  Insecutor  Inscitise  Menstruus,  1914,  ii,  No.  4,  pp. 
58-62. 

2  The  mosquitoes  of  the  Amazon  region.  Ann.  Trop.  Med.  &  ParasitoL,  Liverpool, 
1910,  iv,  pp.  141-150,  pi.  11. 

3  Las  aguas  de  Huacachina.  Informe  presentado  a  la  Sociedad  Geografica  de  Lima, 
1907,  4,  63  pp.,  12  pis.     (Extract  from  Memoria  de  la  Municipalidad  de  Lima,  1906.) 


APPENDIX  213 

which  is  at  the  basal  third,  the  other  two  delimit  white  spots  which  accom- 
pany those  on  the  costa.  Two  other  less  conspicuous  black  spots  occur 
on  the  thu-d  vein  and  upper  branch  of  the  fifth.  The  one  on  the  third  vein 
lies  exactly  beneath  the  costal  white  spot;  the  one  on  the  upper  branch  of 
the  fifth  vein  is  interrupted  at  the  basal  cross-vein. 

It  will  be  seen  that  the  spots  on  the  wing-veins,  as  far  as  indicated, 
correspond  with  those  of  Anopheles  pseudopimctipennis.  Black  spots 
existmg  in  the  latter  species  on  the  second,  fourth  and  sixth  veins,  as  well 
as  small  spots  near  the  base  of  the  fifth  vein  and  the  tip  of  its  lower  branch 
are  not  indicated.  Yet  it  seems  certain  that  these  discrepancies  are  due  to 
omissions  or  oversight,  or,  still  more  likely,  damaged  condition  of  the  speci- 
mens, rather  than  to  any  existing  differences. 

This  opinion  is  substantiated  by  a  specimen  declared  to  be  identical 
with  Anopheles  peruvianus  and  presented  to  Professor  Brues  by  Dr. 
Gastiaburu.  As  we  learn  from  a  footnote  on  page  xxxvii  of  the  treatise  of 
Tamayo  and  Garcia,  Gastiaburu  had  also  considered  the  species  as  new 
and  had  proposed  to  describe  it  as  Anopheles  multiniaculaiis  but  in  his 
thesis  had  suppressed  this  name.  The  specimen  given  to  Professor  Brues 
is  the  one  that  formed  the  basis  of  Gastiaburu's  A.  multimaculatis,  later 
declared  to  be  identical  with  .4.  peruvianus.  It  is  a  male  with  the  spots 
on  the  wing-veins  somewhat  obliterated  in  the  posterior  region  by  loss  of 
scales.  However,  by  careful  scrutiny  the  distribution  of  black  and  white 
scales  can  be  traced  and  is  found  to  agree  perfectly  with  pseudopuncti- 
pennis.  As  to  the  pale  rings  stated  to  exist  at  the  bases  of  the  tarsal  joints 
in  A.  peruvianus,  these  are  merely  due  to  reflection  of  light  and  not  to  any 
difference  in  the  color  of  the  scales  at  those  points.  The  ornamentation  of 
the  palpi  as  described  for  the  female  A .  peruvianus  corresponds  with  typical 
pseudopunctipennis. 

Anopheles  peruvianus  is  based  on  specimens  from  Lake  Huacachina. 
This  lake  is  near  lea,  south  of  Lima  and  not  far  from  the  coast,  at  an  alti- 
tude of  about  1,200  feet.  Tamayo  states  that  A.  peruvianus  is  common  in 
different  parts  of  the  coast  region  and  that  it  occurs  also  in  the  montana, 
that  is,  in  the  forest  region  on  the  eastern  slopes  of  the  Andes.  The  follow- 
ing localities  are  specifically  mentioned.  West  of  the  continental  divide, 
Huacachina,  San  Pedro  de  Lloc  and  Lima  and  its  environs;  on  the  eastern 
slopes  of  the  Andes,  Chanchamayo.  It  should  be  explained  that  this  last 
localit}'  is  not  strictly  within  the  humid  forest  region,  but  rather  in  a  partly 
open  transition  zone.  Gastiaburu's  specimen  probably  came  from  the 
vicinity  of  Lima.  Before  me  are  two  other  specimens  of  A.  pseudopuncti- 
pennis collected  by  Townsend  in  the  region  about  Lima,  one  at  San 
Bartolome,  altitude  about  4,900  feet,  the  other  in  Verrugas  Canyon,  at 
about  5,500  feet  altitude. 

When  we  consider  the  distribution  of  Anopheles  pseudopunctipennis, 
its  occurrence  in  the  Andean  and  coast  regions  of  Peru  appears  in  no  wise 


214  APPENDIX 

remarkable.  It  is  more  especially  the  common  form  of  Anopheles  of  the 
semi-arid  American  tropics.  It  occurs  from  California  and  south-western 
Texas  southward  to  northern  and  north-central  Argentina,  but  within  this 
territory  its  distribution  is  governed  by  local  conditions.  Collections  from 
Mexico  and  Central  America  show  that  it  occurs  in  the  more  arid  portions 
and  is  absent  from  the  humid  forest  zones.  It  is  common  in  Panama. 
In  South  America  it  has  so  far  been  reported  from  only  a  few  widely 
separated  localities.  The  United  States  National  Museum  has  received 
specimens  through  Dr.  J.  H.  Egbert  from  Santa  Marta  on  the  coast  of 
Colombia,  a  distinctly  arid  locality.  Recently  the  species  has  been  found 
in  Trinidad  (near  Port  of  Spain)  by  Professor  F.  W.  Urich.  The  Peruvian 
localities  have  been  already  indicated.  Finally,  the  species  has  been  re- 
ported from  northern  Argentina  (Provinces  of  Jujuy,  Salta,  Tucuman  and 
Santiago  del  Estero),  again  a  semi-arid  region. 

Turning  now  to  the  second  mosquito  described  by  Tamayo  and 
Garcia,  Culex  raymondii,  its  identification  must  be  purely  inferential. 
Neither  the  description  nor  the  figures  offer  details  that  could  lead  to  an 
exact  diagnosis.  The  figure  of  the  larva  shows  by  the  somewhat  elongate 
breathing  tube  with  several  hair  tufts,  together  with  the  large  antennae 
with  large,  outwardly  situated  tuft,  that  the  insect  belongs  to  Culex  in  the 
restricted  sense;  but  beyond  this  the  crude  figure  does  not  permit  one  to 
go.  The  female  is  described  as  having  a  dark  proboscis  and  palpi,  black 
erect  forked  scales  on  the  occiput,  the  recumbent  scales  yellowish  white. 
Mesonotum  without  ornamentation.  Abdomen  dorsally  blackish,  the 
segments  with  basal  white  bands;  venter  pale.  Legs  dark,  the  tarsi  with 
whitish  basal  bands,  most  distinct  on  the  first  j oint.  Claws  simple.  Wings 
unspotted,  the  scales  uniformly  dark  colored.  Total  length  (including 
proboscis)  8.5  mm.  The  larvae  are  said  to  occur  in  unused  wells  in  the 
vicinity  of  Lake  Huacachina. 

There  is  every  probability  that  Culex  raymondii  is  nothing  but  the 
widely  distributed,  semi-domestic  Culex  quinquefasdatus  (  =  fatigans). 
The  description  fits  it  very  well,  as  far  as  it  goes,  with  the  exception  of  the 
ringed  tarsi.  In  the  figures  of  the  two  sexes  the  tarsi  show  no  rings,  and  it 
is  safe  to  assume  that  they  are  as  little  in  evidence  in  this  form  as  in  the 
Anopheles  peruvianus  already  discussed.  Culex  raymondii  is  said  to  occur 
in  Lima  and  its  outskirts,  and  this  again  points  to  our  widely  spread  species 
of  domestic  habits,  Culex  quinquefasdatus,  as  do  the  larval  habits  above 
indicated. 

Following  is  an  annotated  list  of  the  mosquitoes  now  known  to  occur  in 
Peru.  Professor  C.  H.  T.  Townsend,  until  recently  entomologist  to  the 
Peruvian  government,  has  most  liberally  placed  at  my  disposal  his  notes 
on  mosquitoes  and  mosquito  conditions  in  Peru  and  I  have  made  free  use 
of  them.  The  Peruvian  mosquitoes  may  be  grouped  under  four  headings, 
as  follows: 


APPENDIX  215 

1.  Cosmopolitan  species  of  the  tropical  and  subtropical  zones,  occurring 
only  in  association  with  man  and  independent  of  moisture  conditions. 

Culex  quinqiiefasciatus  Say  (C.  fatigans  Auct.) 

Reported  by  Tamayo  and  Garcia  (as  Culex  raymondii)  from  Lima  and  vicinity 
and  Huacachina.  The  following  is  abstracted  from  To\\Tisend's  notes:  "This 
species  has  been  taken  b,v  me  from  Ancon  at  sea  level  to  Chosica  at  2,800  feet 
and  about  the  upper  hmits  of  the  foothill  zone.  It  is  the  common  mosquito  of 
the  whole  region  bordering  the  coast  below  an  elevation  of  about  3,000  feet.  I 
found  it  breeding  in  the  street  wells  of  Ancon,  and  it  is  the  common  mosquito  of 
Lima  and  vicinity.  It  is  not  as  abundant  at  Ancon  as  Aedes  calopus,  and  seems  to 
be  overridden  by  the  latter  along  the  extreme  coast  Une.  It  is  the  only  mosquito 
I  have  observed  at  Chosica.  It  is  comparatively  scarce  in  individuals  both  in 
Lima  and  in  Chosica.  A  recent  epidemic  of  dengue  in  Barranco,  a  suburb  of  Lima, 
occurring  during  the  cooler  season  of  1913,  was  probably  spread  by  this  mosquito." 

Aedes  calopus  Meigen  {Stegomyia  fasciata  Auct.) 

The  following  is  from  Townsend's  notes:  "This  species  extends  all  the  way 
down  the  west  coast  of  South  America  to  the  Antofagasta  region  of  northern  Chile, 
and  doubtless  at  times  much  farther  to  the  south.  I  have  found  this  species  at 
Piura,  which  is  well  inland  but  only  two  hundred  feet  or  less  above  the  sea.  I  have 
found  it  abundant  at  Ancon,  a  beach  resort  an  hour  north  of  Lima  by  train,  where 
it  breeds  in  the  street  wells.  These  wells  are  largely  open,  without  pumps,  or  only 
imperfectly  covered,  allowing  ingress  and  egress  of  the  mosquitoes.  Far  within 
the  rain  forest  region  to  the  east  of  the  Andes,  along  the  navigable  upper  stretches 
of  the  rivers  where  commerce  has  introduced  it,  this  species  also  occurs,  notably  in 
the  Iquitos  region."  The  species  has  been  reported  from  Iquitos  also  by  Newstead 
and  Thomas. 

2.  Coast  species  breeding  in  salt  or  brackish  water. 

Aedes  epinolus  Dyar  and  Knab 

Closely  related  to  Aedes  toeniorhynchus  and  niger  and  perhaps  best  considered  a 
subspecies.  Townsend's  observations  are  as  follows:  "  This  is  the  Peruvian  salt- 
marsh  mosquito  and  conforms  in  general  habits  to  the  typical  form  in  other  parts 
of  America.  It  was  found  breeding  in  large  numbers  during  the  first  week  in 
February  (1914)  in  salt  ponds  immediately  back  of  the  beach  at  Ventanillas,  an 
uninhabited  point  about  nine  miles  south  of  Ancon  and  about  half  way  between 
the  latter  and  Callao.  The  ponds  in  question  vary  from  small  to  large  and  stretch 
along  just  inside  the  beach  line  for  some  miles,  being  formed  by  inroads  of  the  sea 
during  unusually  rough  weather.  This  species  reached  Ancon  in  great  swarms 
during  the  last  week  or  so  of  January,  1914,  and  the  municipahty  called  upon  the 
writer  to  make  an  investigation  of  the  plague.  On  February  3  the  adults  were  still 
to  be  found  in  some  numbers  in  Ancon,  though  rapidly  disappearing,  while  it  was 
determined  that  the  species  was  not  breeding  there.  On  February  4  the  ponds  at 
Ventanillas  were  visited.  Small  ponds  showed  numerous  large  nearly  full-grown 
larvae,  without  either  small  larvae  or  pupae.  Large  ponds  showed  no  signs  of  larvae. 
Rims  of  cast  pupal  skins  just  above  the  waterline,  stuck  to  the  hard  gravel,  fringed 
the  ponds,  having  been  left  to  dry  as  the  water  evaporated  to  a  lower  level.  The 
adults  were  present  in  swarms  and  very  blood-thirsty.  The  only  blood  supply 
present  in  this  immediate  region  of  the  coast  is  that  furnished  by  the  seafowl  that 
continually  haunt  the  coast  line.     On  January  11,  1914,  the  sea  was  unusually 


216  APPENDIX 

heavy  along  this  region  of  the  coast.  It  evidently  overrode  the  high  natural  break- 
water of  smooth  pebbles  that  forms  the  beach  at  Ventanillas,  thus  producing  a 
great  breeding  area  for  this  mosquito.  As  a  result  immense  swarms  migrated  from 
these  ponds  a  week  or  two  later,  and  these  reached  Ancon  in  force  to  the  great 
dismay  of  the  inhabitants." 

3.  Species  of  the  dry  regions  of  the  mountains  and  coast. 

Anopheles^  pseudopunctipennis  Theobald 

(Sjmonyms:  franciscanus  McCracken,  tucumanus  Lahille,  argentinus  Brethes, 
peruvianus  Tamayo,  multimaculatis  Gastabuni) 
The  known  distribution  has  been  already  indicated  in  the  previous  discussion. 
Townsend  thinks  that  in  Peru  the  species  probably  ranges  considerably  above 
8,000  feet.  Its  occurrence  must  be  more  or  less  local,  depending,  as  it  does,  upon 
the  presence  of  suitable  breeding  places,  such  as  isolated  pools  in  stream-beds, 
water-holes,  abandoned  weUs,  etc. 

Anopheles  species,  near  maculipes  Theobald 

A  single  female  specimen  in  rather  poor  condition  presented  to  Dr.  Brues  by 
Dr.  Gastiabuni,  along  with  the  specimen  of  A.  pseudopunctipennis  already  dis- 
cussed. The  locality  is  uncertain,  but  most  probably  not  remote  from  Lima. 
The  specimen  resembles  A.  maculipes  and  related  species  in  the  coloration  charac- 
ters and  in  the  shape  of  the  wing-scales,  but  differs  in  numerous  details.  It 
probably  represents  an  undescribed  species,  but  it  seems  inadvisable  to  found  a 
species  in  a  difficult  group  on  a  single  poor  specimen  of  uncertain  origin. 

Phalangomyia  debilis  Dyar  and  Knab 
Matucana  (Brues). 

4.  Species  of  the  humid  forest  region  east  of  the  Andes. 

Anopheles  boliviensis  Theobald 

(Synonyms:  lutzii  Theobald,  not  Cruz;  cruzi  Dyar  and  Knab) 

Described  twice  by  Theobald,  first  from  southern  Brazil  and  then  from  Songo, 
BoUvia.  The  latter  locality  is  presumably  on  the  eastern  side  of  the  Andes  and 
within  the  forest  region.  This  species  breeds  almost  exclusively  in  water  held  by 
the  leaves  of  epiphytic  bromehads  and  therefore  is  a  typical  forest  inhabitant. 
Townsend  says:  "  I  took  this  species  in  the  montana  of  the  province  of  Jaen,  in 
northern  Peru,  from  the  Rio  Charape  at  about  4,500  feet  to  the  lower  slopes  of 
Huascaray  ridge  at  about  7,000  to  7,500  feet.  It  was  abundant  at  both  locahties 
and  an  active  biter.  Both  localities  are  forested,  the  Huascaray  slopes  in  patches, 
the  Rio  Charape  more  thickly.  Both  afford  tree  holes  and  epiphytes  as  breeding 
places,  but  do  not  furnish  other  standing  water,  the  streams  being  comparatively 
swift." 

Anopheles  tarsimaculatus  Goeldi  (cilbimanus  Newstead  and  Thomas) 

Reported  by  Newstead  and  Thomas  as  the  common  Anopheles  at  Iquitos.  This 
is  a  geographic  race  or  subspecies  of  A.  albimanus,  distinguishable  by  the  difference 
in  the  palpal  banding.  It  is  the  chief  malaria  transmitter  in  the  Amazon  region 
and  throughout  most  of  the  Brazil-Guiana  forest  zone. 


APPENDIX  217 

Janthinosoma  posticata  Wiedemann  (musica  Newstead  and  Thomas) 

Reported  from  Iquitos  by  Newstead  and  Thomas.  A  very  widely  distributed 
species  east  of  the  Andes. 

Aedes  leucomelas  Lutz 

Townsend  took  a  single  female  of  this  species  at  Hacienda  Charape  on  the  Rio 
Tabaconas,  about  3,500  to  4,000  feet,  Sept.  18,  1911. 

Mansonia  titillans  Walker 

Iquitos  (Newstead  and  Thomas).  Widely  distributed  through  the  American 
moist  tropics,  but  owing  to  the  peculiar  larval  habits  local  in  occurrence.  The 
larvae  inhabit  permanent  waters  and  live  attached  to  the  roots  of  certain  aquatic 
plants  from  which  they  obtain  the  needed  air.  The  female  is  an  aggressive  blood- 
sucker and  when  abundant  a  serious  pest. 

Mansonia  pseudotitillans  Theobald 

A  single  specimen  from  Iquitos  doubtfully  so  identified  by  Newstead  and 
Thomas. 

Cidex  chrysothorax  Newstead  and  Thomas 

{Neomelaniconion  chnjsothorax  Newstead  and  Thomas,  not  Melanoconion  chryso- 
thorax Peryassii) 

Described  from  Iquitos,  Peru,  and  Mandos,  Brazil.  The  specific  name  is  pre- 
occupied by  the  species  of  Peryassu,  as  the  two  species  are  congeneric  and  belong 
in  Culex.  1  refrain  from  proposing  a  new  name,  as  the  species  may  be  identical 
with  others  already  described. 

Limatus  durhami  Theobald 
Reported  from  Iquitos  by  Newstead  and  Thomas. 

Sabethes  species 

Townsend  has  the  following  note  on  a  species  of  this  genus,  the  members 
of  which  are  strictly  forest  insects.  "  I  took  two  specimens  on  February  12,  1910, 
at  Yahuarmayo  on  the  Rio  Inambari,  about  1,700  feet,  in  the  southern  montana 
of  Peru.  The  species  is  of  a  briUiant  metalUc  coloring,  with  feathered  legs.  It 
was  determined  by  Dr.  A.  Lutz  as  this  genus,  with  the  note:  '  Very  much  Uke  a 
species  I  brought  from  the  Amazons  region.'  " 


218  APPENDIX 

IV.    HETEROPTERA  FROM  THE  WEST  COAST  OF  SOUTH  AMERICA 
By  J.  R.  DE  LA  Torre  Bueno 

The  few  species  collected  in  Ecuador  and  Peru  in  1913,  by  the 
expedition  from  the  School  of  Tropical  Medicine  of  the  Harvard  Medical 
School  have  been  submitted  to  me  for  determination.  All,  except  one, 
were  taken  at  Guayaquil  in  Ecuador;  nine  of  the  thirteen  Ecuadorian 
species  are  new  records  for  that  country,  and  three  considerably  extend 
the  range  of  North  and  Central  American  species.  The  Peruvian  species, 
Nysius  spurcus  (Stal)  is  also  a  new  record  and  greatly  enlarges  its  area 
of  dispersal.  On  the  whole,  small  as  is  the  number  of  species  listed,  it 
contributes  materially  to  our  knowledge  of  distribution. 

The  arrangement  of  the  Cimicids  (Pentatomids)  is  according  to  Kir- 
kaldy;  the  remaining  families  are  arranged  according  to  Lethierry  and 
Severin's  Catalogue  and  to  Bergroth's  supplements  thereto. 

Thyanta  antigiiensis  Westw. 

There  is  one  specimen  only  of  this  small  species,  which  has  seemingly 
not  been  heretofore  recorded  from  South  America;  the  Southwestern  United 
States,  Mexico,  Central  America  and  the  Antilles  being  the  known  range. 

Symphylus  deplanatus  H.  S. 

This  species  has  not  been  recorded  heretofore  from  further  south  than 
Panama.  This  would  seem  to  be  its  first  reported  appearance  on  the 
Pacific  coast  of  South  America,  where  one  specimen  was  secured. 

Pachylis  laticornis  Fabr. 

Only  one  specimen  of  this  wide-spread  and  common  form  was  obtained. 
It  has  been  recognized  heretofore  from  Costa  Rica  to  the  Argentine  Re- 
public, including  Ecuador,  in  the  Transandean  region,  but  not  from  the 
Pacific  side. 

Capaneus  ohscuratus  Mont. 

One  typical  9  specimen  was  collected  at  Guayaquil,  the  type  locality. 

Machtima  crucigera  Fabr. 

A  <?  and  a  9  are  present,  of  this  unmistakable  species,  which  would 
seem  to  be  known  from  Brazil  only,  so  far  as  published  records  available 
are  concerned.     This  is  a  new  record  and  remarkably  extends  its  range. 

Hypselonotus  lineatus  Stal 

The  ten  specimens  sent  are  the  first  to  be  known  from  the  Pacijfic  coast 
of  South  America.     The  species,  described  from  Mexico,  has  also  been 


APPENDIX  219 

secured  in  Honduras,  on  the  Atlantic  (or  rather,  Gulf)  side  of  Central 
America. 

Harmostes  reflexulus  Fabr. 

There  is  one  typical  specimen  of  this  species  in  the  collection,  first  record 
from  Ecuador,  although  previously  known  from  such  widely  separated 
lands  as  :\Iexico  and  the  Argentine  Republic,  but  from  none  of  the  inter- 
vening countries. 

Rhopalus  rufescens  Spinola 

This  species  hitherto  known  only  from  Chile,  whence  it  was  first  de- 
scribed, is  represented  by  one  specimen  from  Guayaquil. 

Rhopalus  sidae  Fabr. 

There  are  two  Guayaquil  specimens  of  this  common  form  in  the  collec- 
tion, a  first  record,  although  it  is  said  to  be  distributed  from  Mexico  to 
Argentine  Republic. 

Dysdercus  ruficolUs  Linne 

This  abundant  and  wide-spread  form  is  represented  by  nine  specimens. 
It  is  known  from  all  South  America  (except  Peru  and  Chile)  and  from 
Mexico  as  well. 

Nysius  spu7xus  Stal 

The  one  specimen  present  from  San  Bartolome  represents  the  Hemip- 
terous  fauna  of  Peru.  It  is  of  interest,  as  heretofore  it  has  been  known 
only  from  Mexico,  Honduras  and  Brazil. 

Melanolestes  morio  Er. 

This  darkling  Reduviid  is  represented  by  one  specimen  from  Guayaquil, 
seemingly  the  first  Ecuadorian  record.  Its  previous  records  are  from 
Colombia,  the  Guianas  and  Brazil. 

Zelus  (Diplodus)  sp. 

In  the  collection  there  are  7  specimens  of  an  undetermined  species  from 
Guaj^aquil.  This  genus  is  sadly  in  need  of  a  careful  revision,  especially  of 
the  tropical  American  forms. 

Belostoma  asiaticum  Mayr. 

Nine  specimens  are  present.  Apparently  this  species  heretofore  was 
unknown  from  Ecuador,  although  recorded  from  Peru  and  Chile  on  the 
Pacific  coast,  as  well  as  from  Brazil,  Mexico  and  Argentine  Republic. 


220  APPENDIX 


V.    LIST  OF  THE  BEES  OBTAINED  BY  THE  EXPEDITION 

The  present  list  is  taken  from  a  paper  by  Professor  T.  D.  A.  Cockerell/ 
who  kindly  examined  all  of  the  bees  that  were  collected. 

Xylocopa  brasilianorum,  subsp.  bruesi  CkU.     San  Bartolome. 
Xylocopa  transitoria  Perez.     Guayaquil. 
Eulaema  bruesi  Ckll.     Guayaquil. 
Bombus  coccineus  Friese.     Matucana. 
Melipona  mimetica  Cldl.     Guayaquil. 
Trigona  leucogastra  Cldl.     Guayaquil. 
Megachile  pyrrhogastra  Ckll.     Guayaquil. 
Megachile  philinca  Ckll.     Guayaquil. 
Megachile  garleppi  Friese.     San  Bartolome. 
Megachile  ecuadoria  Friese.     Matucana. 
Coelioxya  haematura  Ckll.     Guayaquil. 
Coelioxys  leucochrysea  CkU.     Guayaquil. 
Coelioxys  rufibasis  Ckll.     Guayaquil. 
Coelioxys  tumerifora  Ckll.     San  Bartolome. 
Hypaiithidium  ecuadorium  Friese.     Guayaquil. 
Anthidium  22-pimctatuni  Friese.     Guayaquil. 
Anthidium  matucanense  Ckll.     Matucana. 
Triepeolus  megadelphus  Ckll.     Guayaquil. 
Centris  nitida,  subsp.  geminata  Ckll.     Guayaquil. 
Melitoma  euglossoides  Lep.  &  Serv.     Guayaquil. 
Xenoglossa  citrullina  Ckll.     Guayaquil. 
Florilegus  pavoninus  Ckll.     Guayaquil. 
Florilegus  purpurascens  Ckll     Guayaquil. 
Melissodes  ecuadoria  Bertoni  &  Schrottky.     Guayaquil. 
Leptometria  pacifica  Ckll.     Guayaquil. 
Chalepogenus  alfkeni  Ckll.     Guayaquil. 
Exomalopsis  zexmeniae  Ckll.     San  Bartolome. 
Exomalopsis  bruesi  Cldl.     Guayaquil;  San  Bartolome. 
"     Agapostemon  nasutus  Smith.     San  Bartolome. 
Augochlora  binghami  Ckll.     Guayaquil. 
Augochlora  metallica  Fabr.     Guayaquil. 
Augochlora  thalia  Smith.     Guayaquil. 
Augochlora  cladopyga  Ckll.     Guayaquil. 
Augochlora  matucanensis  CkU.     Matucana. 
Lonchopria  inca  Ckll.     Matucana. 
Colletes  miminca  CkU.     Matucana. 

1  J.  New  York  Entom.  Soc,  1914,  xxii,  pp.  306-328. 


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